MHC class II antigen-presenting systems and methods for activating CD4+ T cells

ABSTRACT

The present invention relates to synthetic antigen-presenting matrices, their methods of making and their methods of use. One such matrix is cells that have been transfected to produce MHC antigen-presenting molecules with one or more accessory molecules. The matrices are used to activate naive CD4 +  T cells as well as shift the ongoing activation state into a preferred differentiated population of either Th1 or Th2 cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 09/715,891filed Nov. 17, 2000, pending, which is a divisional of U.S. applicationSer. No. 09/194,285 filed Apr. 12, 1999 now U.S. Pat. No. 6,355,479, andis a divisional of U.S. application Ser. No. 10/822,173 filed Apr. 8,2004, pending, which is a continuation of 09/715,231 filed Nov. 17,2000, now abandoned, which is a divisional of U.S. application Ser. No.09/194,285 filed Apr. 12, 1999 now U.S. Pat. No. 6,355,479, which is arule 371 continuation of application Ser. No. PCT/US97/08697, filed May22, 1997, which is a continuation-in-part application of U.S.provisional application Ser. No. 60/018,175 filed May 23, 1996, nowabandoned.

TECHNICAL FIELD

The present invention relates to materials and methods of activatingCD4⁺ T cells with specificity for particular antigenic peptides, the useof activated T cells in vivo for the treatment of a variety of diseaseconditions, and compositions appropriate for these uses.

BACKGROUND

Though the T cell repertoire is largely shaped during T cell developmentin the thymus, mature CD4⁺ T cells are also regulated extrathymically.Whereas, some conditions of activation lead to tolerance reflectingeither anergy or clonal elimination, other conditions lead to a changein the type of response observed. For CD4⁺ T cells, this change infunctional phenotype is largely a change in the pattern of cytokinesproduced. Although CD4⁺ T cells that are subject to acute activationmaintain the ability to produce multiple cytokines, T cells obtainedunder conditions of chronic stimulation frequently demonstrate a morerestricted pattern of cytokine production. For example, T cell clonesmaintained by repeated stimulation in vitro have defined two majorfunctional categories of CD4⁺ T cells referred to as Th1 and Th2 typecells. Th1 type cells produce primarily interleukin-2 (IL-2),interferon-γ (IFN-γ) and tumor necrosis factor (TNF), all of which arereferred to as inflammatory cytokines. In contrast, Th2 type cellstypically produce IL-4, IL-5, and IL-10 and are important for antibodyproduction and for regulating the responses of Th1 type cells.

Although such extreme segregation in cytokine production is often notseen during in vivo T cell responses, recovery from certain types ofinfections, such as Leishmania, is associated with preferentialproduction of IL-2/IFN-γ. Mice that mount a Th2 response to Leishmaniafail to contain the infection and ultimately die. Inappropriateproduction of cytokines of the Th2 type response has been frequentlylinked to allergic type diseases such as asthma and contact sensitivity.For review on activation of CD4⁺ T cells and role in allergic disease,see Hetzel and Lamb, Clinical Immunol. Immunopath., 73:1-10 (1994).

Perhaps the strongest association of human disease with skewed patternsof cytokine production is the association of Th1 responses and Th1 typecytokines with autoimmune disease. Strong evidence in experimentalmodels indicates that many types of autoimmunity including diabetes,experimental models for multiple sclerosis, autoimmune thyroiditis, andthe like are mediated by Th1 type CD4⁺ T cells. The expression ofTh2-associated cytokines, such as IL-4, in these models interfere withthe development of autoimmune disease. Th2 type cytokines dampen theresponse of Th1 type cells while the Th1 type cytokines antagonize thedevelopment of Th2 type responses.

In view of the association of particular activated T cell subsets withparticular disease conditions, a need therefore exists to be able todirect the proliferation and activation of CD4⁺ T cells to a desired Tcells subset, a process that is extremely beneficial in altering thecourse of disease. One potential solution is to activate in vitro CD4⁺ Tcells that are first isolated from a subject who may optionally behaving either allergy or autoimmune conditions to produce cellssecreting a preferred cytokine profile. The resultant activated T cellsare then reintroduced to the subject to alter the course of disease andperhaps even provide a long term cure.

The challenge in this approach, now solved by the present invention, isthe difficulty in defining activation conditions that reproduciblygenerate CD4⁺ T cell subsets that produce the desired therapeuticcytokine profile. Expression of particular cytokines is linked to aparticular antigen presenting cell (APC) and their associated accessory(assisting) molecules. For a review of the surface proteins serving asaccessory molecules that are involved in T cell costimulation, seeMondino and Jenkins, J. Leukocyte Biol., 55:805-815 (1994). Since boththe cytokines produced by the APC and the coordinately expressedaccessory molecules are themselves regulated by multiple factors,including the type of antigen, the affinity of the T cell receptor(TCR)-antigen interaction, antigen concentration and the like,predicting the outcome of T cell activation upon antigen presentation ishistorically very difficult. Indeed, as additional accessory moleculeshave been proposed for the activation process in vivo, it has becomeincreasingly clear that many diverse molecules are involved in theregulation of T cell responses and act in combinatorial fashion toeffect the outcome of T cell activation.

Prior to the present invention, the co-expression of selected MHC classII molecules in conjunction with one or more selected accessorymolecules has not been possible. The present invention now presents asolution to predictably generate a preferred T cell phenotype throughthe reproducible activation of T cells to generate either Th1 or Th2type T cells. The invention describes the generation of synthetic APCthat present, in a neutral background, MHC class II molecules incombination with defined accessory molecules. The MHC class II moleculesand defined accessory molecules are expressed in a nonmammalian insectcell and can be presented in a variety of forms of synthetic APCincluding insect cells displaying the molecules.

The advantage of using the insect cells as the expression andpresentation vehicles for the MHC class II/accessory moleculecompositions of this invention is that the cells do not endogenouslyproduce regulatory cytokines and do not express mammalian accessorymolecules. This overcomes the inherent unpredictability of usingmammalian APC that express many molecules that are capable of alteringthe T cell response. In addition, the insect cell expression systemdescribed in the present invention provides for the expression of MHCclass II molecules without bound peptide (i.e., “empty” molecules) thatcan be produced under certain restrictive circumstances, such astemperature requirements. At physiological temperatures, these “empty”molecules are normally unable to reach the cell surface as class IIwithout bound peptide are very thermolabile. The invention utilizes thecapacity of “empty” MHC class II compositions to allow for the exogenousloading of selected peptides along with the ability to providedendogenously loaded counterparts.

A recombinant glycosyl-phosphatidylinositol (GPI)-modified MHC class Imolecule (HLA-A2.1:GPI/β₂m) was generated in the above-described insectcell system to produce antigen presenting cells as described inInternational Publication Number WO 96/12009 by Tykocinski. In thatpublication, the recombinant GPI-modified MHC class I molecules areisolated from the insect cell by affinity purification for subsequentreincorporation into cell membranes. In other aspects, the publicationdescribes the preparation of a GPI-modified MHC class I moleculeco-anchored on a cell membrane with a GPI-modified B7.1 costimulatorymolecule. Although the publication states that GPI-modified MHC class IImolecules can be prepared as described for those of MHC class I, thepublication does not present any details for such preparation.

In contrast, the present invention provides and describes a unique meansbased on the co-expression of a selected MHC class II haplotype inconjunction with one or more accessory molecules, such as B7.1, toactivate CD4⁺ T cells resulting in the differentiation to a particular Tcell subset, Th1 or Th2 cells, that effect a preferred cytokine profileinfluence. The invention provides the advantage of selectivelyactivating CD4⁺ T cells in vitro to a preferred T cell subset thereafterallowing for the reintroduction of the activated T cells into thepatient. The present invention thus provides the ability to combineindividual presenting molecules with particular accessory molecules forexpression in selected combinations that permits reproducibility andpredictability for selectively activating CD4⁺ T cells to a desired Tcell subset not available in other approaches.

BRIEF SUMMARY OF THE INVENTION

It has now been discovered that recombinant MHC class II moleculesexpressed in combination with selected accessory molecules, includingcostimulatory molecules and adhesion molecules, are effective inactivating CD4⁺ T cells to become armed effector T cells that recognizetarget cells on which MHC class II heterodimer is expressed forcomplexation with peptide. Activation is characterized by proliferationand differentiation into effector T cell subsets, Th1 and Th2, thatsecrete particular cytokines. Th1 and Th2 type T cells are respectivelyreferred to as inflammatory cells and T-helper cells.

Thus, the present invention relates to a synthetic antigen presentingsystem, also referred to as APC, for producing and presenting amammalian, preferably human, MHC class II molecule in combination withone or more accessory molecules to activate CD4⁺ T cells.

In one embodiment, the system relates to a synthetic antigen presentingmatrix having a support and at least the extracellular portion of a MHCclass II heterodimeric molecule operably linked to the support andcapable of binding to a selected peptide. The matrix also includes anaccessory molecule operably linked to the support. The accessorymolecule interacts with a specific receptor present on the CD4⁺ T cell.The MHC class II and accessory molecules are present in sufficientnumbers to activate a population of CD4⁺ T cells specific for the MHCclass II/peptide combination when the peptide is bound to theextracellular portion of the MHC molecule.

It has been found that an antigen presenting matrix having both a MHCclass II heterodimer or at least the extracellular portion thereofloaded with a peptide specific for that MHC, together with an accessorymolecule, provides a synergistic reaction in activating CD4⁺ T cells.Examples of accessory molecules are costimulatory molecules, includingB7.1 and B7.2, adhesion molecules such as intercellular cell adhesionmolecule-1 (ICAM-1) and lymphocyte function-associated antigen-3(LFA-3), and survival molecules such as Fas ligand (FasL) and CD70. Insome embodiments, the extracellular portion of such accessory moleculescan also be used in the present invention.

The support used for the matrix can take several different forms.Examples for the support include solid support such as metals orplastics, porous materials such as resin or modified cellulose columns,microbeads, microtiter plates, red blood cells and liposomes.

Another type of support is a cell fragment, such as a cell membranefragment. An entire cell is also contemplated as a support. In thisembodiment, the matrix is actually cells which have been transformedwith one or more expression vectors containing genes for the expressionof MHC class II α- and β-chains along with at least one accessorymolecule. The expressed proteins are then transported to the cellmembrane where the transmembrane domain of the class II chains provideanchors allowing the extracellular domain to be displayed on the outercell surface, thereby creating a synthetic antigen presenting cell(APC). The expression vectors contain the selected genes, preferably inthe form of a cDNA sequence, operably linked to a promoter that iseither constitutive or inducible.

The MHC α- and β-chains associate together forming a MHC class IIheterodimer which binds to a peptide specific for that heterodimer. Withthe present invention, two methods of loading peptides onto MHC class IIheterodimers are contemplated. In one embodiment, the peptide is loadedintracellularly following proteolytic processing of internalized intactprotein into peptide fragments. The peptides are then loaded onto newlygenerated MHC class II molecules while they are still within the cell.Alternatively, the MHC class II molecules are expressed as emptymolecules on the cell surface and synthetic or processed peptidereagents are then loaded extracellularly onto the MHC class IIheterodimer.

Nucleotide sequences for encoding at least one accessory molecule geneoperably linked to a promoter in a vector are also introduced into thecell. Following expression, the accessory molecule is coordinatelyanchored on the surface of the cell along with the MHC class IIheterodimer in sufficient numbers to activate a population of CD4⁺ Tcells lymphocytes specific for the MHC class II/peptide complex. Othermolecules referred to as antigen processing assisting molecules are alsocontemplated for use in generating recombinant APC. These molecules areeither provided by the cell used as APC or exogenously through anexpression vector system as described above. Examples of such antigenprocessing assisting molecules include invariant chain, lysosomalenzymes and H2-M and H2-O molecules.

The cell line is synthetic in that at least one of the genes describedabove is not naturally present in the cells from which the cell line isderived. It is preferable to use a poikilotherm cell line because MHCmolecules are thermolabile. A range of species are useful for thispurpose. See, for example, U.S. Pat. No. 5,314,813 to Petersen et al.which discusses numerous species for this use, the disclosure of whichis hereby incorporated by reference. Eukaryotic cells and preferablyinsect cells are used as APC. Preferred insect cells include Drosophila(fruit fly) and Spodoptera (butterfly).

MHC class II molecules have been expressed in insect cells such asDrosophila and Spodoptera cells. Since these cells do not have all thecomponents of a mammalian immune system, the various proteins involvedin the peptide loading machinery are absent from such cells. The lack ofmammalian peptide-loading machinery allows the introduced mammalian MHCclass II molecules to be expressed as empty molecules at the cellsurface when the cells are cultured at thermostabile temperaturerestrictive conditions, such as at 28° C. In contrast, at 37° C., emptyClass I molecules are thermolabile and tend to disintegrate. Thus, byincubating MHC class II-expressing Drosophila cells with peptides thatspecifically bind to anchored MHC class II molecule, virtually everyclass II molecule is loaded with one and the same peptide. Moreover, theinvention provides for the means to introduce any known MHC class II α-and β-chain genes into an expression vector thereby overcoming theinherent limit to the number of MHC class II molecules expressed in anyone mammal.

In the present invention, a specifically effective synergistic reactionin driving CD4⁺ T cells to a Th1-type response characterized by anincrease in the cytokine interleukin-2 (IL-2) results from a Drosophilaantigen presenting cell having MHC class II molecules bound with apeptide, a costimulatory molecule, and an adhesion molecule. Inparticular, a highly effective synergistic generation of IL-2 productioncoupled with CD4⁺ proliferation results from the combination of B7.2 andICAM-1. In contrast, without ICAM-1 but with either B7.1 or B7.2, theDrosophila APC system loaded with peptide induced a Th2-type responsecharacterized by an increase in IL-4. Thus, ICAM-1 antagonized theTh2-type response resulting in a Th1-type phenotype.

A Th1 phenotype characterized by IL-2 production coupled withproliferative responses also resulted from a synthetic antigenpresenting cell having CD70 expressed simultaneously with ICAM-1 with orwithout B7.2.

Therefore, the selection of MHC class II genes in combination with atleast one accessory molecule genes for expression thereof in an APC ofthis invention can be tailored depending upon the desired outcome foreffecting proliferation and phenotypic activation of CD4⁺ T cells.

The present invention also relates to methods for making the syntheticAPC systems as described above in which at least one expression vectorcontaining genes for a MHC class II heterodimer and an accessorymolecule is introduced.

Methods of producing activated CD4⁺ T cells in vitro are alsocontemplated. One preferred method comprises contacting, in vitro, CD4⁺cells with a synthetic MHC class II/accessory molecule-bearing APCdescribed above for a time period sufficient to activate, in anantigen-specific manner, a population of CD4⁺ T cells. The method mayfurther comprise (1) separating the activated CD4⁺ cells from theantigen-presenting matrix; (2) suspending the activated CD4⁺ cells in anacceptable carrier or excipient; and (3) administering the suspension toan individual in need of treatment. As previously discussed, theantigens may comprise native or undegraded proteins or polypeptides, orthey may comprise antigenic polypeptides which have been cleaved orsynthesized into peptide fragments comprising at least 8 amino acidresidues prior to incubation with the mammalian MHC class IIheterodimeric molecules.

In addition to the utility of being able to direct the activation ofCD4⁺ T cells to a desired T cell subset as described above, the abilityto express any MHC class II molecule provides the means to identifyunknown CD4⁺-activating peptide specific for that particular MHC classII molecule. As such, the present invention contemplates the activationof CD4⁺ T cells through the screening of a peptide library withsynthetic APC expressing a particular MHC class II heterodimer.

In a further embodiment, the synthetic APC system described herein isuseful for isolation of reactive CD4⁺ T cells from a heterologouspopulation of cells. Such isolation provides the ability to monitorongoing CD4⁺ T cell-mediated responses in disease conditions in apatient.

In another variation of the above, in view of the ability to selectivelyactivate CD4⁺ T cells into a particular T cell subset for producing apreferred cytokine profile, the invention relates to methods of treatingconditions in patients mediated by a undesirable CD4⁺ response. Suchdisease conditions characterized by either a Th1- or Th2-type responseinclude autoimmune diseases, allergy and cancer. The therapeutic goal isto introduce CD4⁺ T cells activated to a preferred T cell subset toantagonize an ongoing CD4⁺ T cell response. Thus, the method comprises(1) obtaining a fluid sample containing resting or naive CD4⁺ cells fromthe patient; (2) contacting, in vitro, the CD4⁺ cells with a selectedsynthetic peptide-loaded APC of this invention for a time periodsufficient to activate, in an antigen-specific manner, the CD4⁺ cells;and (3) administering the activated CD4⁺ cells to the patient.

Other embodiments are apparent to one skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C diagram the construction of expression plasmids pRmHa-2 andpRmHa-3. In FIG. 1A, pRmHa-2 construction is shown; in FIG. 1B, pRmHa-3construction is shown; and in FIG. 1C, the pRmHa-3 vector isillustrated, showing the restriction, polylinker, promoter, andpolyadenylation sites, as well as a site at which a nucleotide sequencemay be inserted for expression.

FIG. 2 shows the proliferative response of DO10 T cell receptor (TCR)transgenic mouce cell line (Tg cells) when cultured in the presence ofDrosophila cell lines with or without ovalbumin (OVA) peptide loadedonto the surface-expressed MHC class II heterodimer. Proliferation isassayed as described in Example 5. The proliferation is measured incounts per minute (cpm)×1000 as plotted on the Y-axis against increasingconcentrations of OVA peptide in μM on the X-axis. T-S (diamond markedline) shows responses with control splenic APC. Proliferative responseswith recombinant MHC class II alone are shown in the line having closedcircles. Those with MHC class II combined with either costimulatorymolecules B7.1 or B7.2 are shown respectively with lines having open andclosed squares.

FIG. 3 shows proliferative responses to recombinant MHC class II alone(closed circle line), with MHC class II plus B7.2 (closed square line),with MHC class II plus ICAM-1 (open triangle line) and with MHC class IIplus B7.2 and ICAM-1 (open diamond line). Refer to FIG. 2 legend forother details.

FIGS. 4A-4D show the cytokine profile produced in response to activationof CD4⁺ T cells when cultured in the presence of Drosophila APC havingrecombinant MHC class II alone or in combination with B7.1 or B7.2costimulatory molecules. Splenic APC (labeled T-S) are control assays.The assays are performed as described in Example 5. FIGS. 4A-4Drespectively show the cytokines Il-2, Il-4, IFN-γ and Il-10 in ng/ml asplotted on the Y-axis. Cytokine profiles were assessed over three daysbetween day 3 and 5 of culture.

FIGS. 5A-5D show the cytokine profile produced in response to activationof CD4⁺ T cells when cultured in the presence of Drosophila APC havingrecombinant MHC class II alone or in combination with B7.2 costimulatorymolecule, ICAM-1 and with B7.2 and ICAM-1. FIGS. 5A-5D respectively showthe cytokines Il-2, Il-4, IFN-γ and 1′-10 in ng/ml as plotted on theY-axis. Refer to FIG. 4 legend for other details.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

Amino Acid Residue: An amino acid formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues described herein are preferably in the “L” isomeric form.However, residues in the “D” isomeric form can be substituted for anyL-amino acid residue, as long as the desired functional property isretained by the polypeptide. NH₂ refers to the free amino group presentat the amino terminus of a polypeptide. COOH refers to the free carboxygroup present at the carboxy terminus of a polypeptide. In keeping withstandard polypeptide nomenclature (described in J. Biol. Chem.,243:3552-3559 (1969) and adopted at 37 CFR §1.822(b) (2)), abbreviationsfor amino acid residues are shown in the following Table ofCorrespondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp tryptophan R Arg arginine DAsp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine XXaa Unknown or other

It should be noted that all amino acid residue sequences representedherein by formulae have a left- to-right orientation in the conventionaldirection of amino terminus to carboxy terminus. In addition, the phrase“amino acid residue” is broadly defined to include the amino acidslisted in the Table of Correspondence and modified and unusual aminoacids, such as those listed in 37 CFR 1.822(b)(4), and incorporatedherein by reference. Furthermore, it should be noted that a dash at thebeginning or end of an amino acid residue sequence indicates a peptidebond to a further sequence of one or more amino acid residues or acovalent bond to an amino-terminal group such as NH₂ or acetyl or to acarboxy-terminal group such as COOH.

Recombinant DNA (rDNA) molecule: A DNA molecule produced by operativelylinking two DNA segments. Thus, a recombinant DNA molecule is a hybridDNA molecule comprising at least two nucleotide sequences not normallyfound together in nature. rDNA's not having a common biological origin,i.e., evolutionarily different, are said to be “heterologous”.

Vector: A rDNA molecule capable of autonomous replication in a cell andto which a DNA segment, e.g., gene or polynucleotide, can be operativelylinked so as to bring about replication of the attached segment. Vectorscapable of directing the expression of genes encoding for one or morepolypeptides are referred to herein as “expression vectors”.

Upstream: In the direction opposite to the direction of DNAtranscription, and therefore going from 5′ to 3′ on the non-codingstrand, or 3′ to 5′ on the mRNA.

Downstream: Further along a DNA sequence in the direction of sequencetranscription or read out, that is traveling in a 3′- to 5′-directionalong the non-coding strand of the DNA or 5′- to 3′-direction along theRNA transcript.

Reading Frame: A particular sequence of contiguous nucleotide triplets(codons) employed in translation that define the structural proteinencoding-portion of a gene, or structural gene. The reading framedepends on the location of the translation initiation codon.

Polypeptide: A linear series of amino acid residues connected to oneanother by peptide bonds between the alpha-amino group and carboxy groupof contiguous amino acid residues.

Protein: A linear series of greater than 50 amino acid residuesconnected one to the other as in a polypeptide.

Receptor: A receptor is a molecule, such as a protein, glycoprotein andthe like, that can specifically (non-randomly) bind to another molecule.

Substantially Purified or Isolated: When used in the context ofpolypeptides or proteins, the terms describe those molecules that havebeen separated from components that naturally accompany them. Typically,a monomeric protein is substantially pure when at least about 60% to 75%of a sample exhibits a single polypeptide backbone. Minor variants orchemical modifications typically share the same polypeptide sequence. Asubstantially purified protein will typically comprise over about 85% to90% of a protein sample, more usually about 95%, and preferably will beover about 99% pure. Protein or polypeptide purity or homogeneity may beindicated by a number of means well known in the art, such aspolyacrylamide gel electrophoresis of a sample, followed byvisualization thereof by staining. For certain purposes, high resolutionis needed and high performance liquid chromatography (HPLC) or a similarmeans for purification utilized.

Synthetic Peptide: A chemically produced chain of amino acid residueslinked together by peptide bonds that is free of naturally occurringproteins and fragments thereof.

B. MHC Class II Heterodimers. Accessory Molecules and Antigen ProcessingAssisting Molecules

The present invention relates to a synthetic antigen-presenting systemfor use in activating CD4⁺ T cells. Though the T cell repertoire islargely shaped during T cell development in the thymus, mature CD4⁺ Tcells are also regulated xtrathymically. Although some conditions ofactivation lead to tolerance reflecting either anergy or clonalelimination, other conditions lead to a change in the type of responseobserved. For CD4⁺ T cells, this change in functional phenotype islargely a change in the pattern of cytokines produced. Although CD4⁺ Tcells that are subject to acute activation maintain the ability toproduce multiple cytokines, T cells obtained under conditions of chronicstimulation frequently demonstrate a more restricted pattern of cytokineproduction. For example, T cell clones maintained by repeatedstimulation in vitro have defined two major functional categories ofCD4⁺ T cells referred to as Th1 and Th2 type cells. Th1 type cellsproduce primarily interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumornecrosis factor (TNF), all of which are referred to as inflammatorycytokines. As such, the TH1 cells are sometimes referred to asinflammatory T cells which then activate macrophages to killintravesicular pathogens they harbor as mediated by peptides generatedfrom processing of pathogenic proteins presented to CD4⁺ T cells. Incontrast, Th2 type cells typically produce IL-4, IL-5, and IL-10 and areimportant for antibody production and for regulating the responses ofTh1 type cells. As such, Th2 cells are sometimes referred to as helper Tcells that activate B cells to make antibody in response to peptidesderived from extracellular pathogens and toxins that are presented toCD4⁺ T cells.

A CD4⁺ T cell is defined as a T cell or a T lymphocyte that has the CD4⁺co-receptor on its cell surface in conjunction with the presence ofeither α:β or γ:Δ heterodimeric receptor associated with the proteins ofthe CD3 complex.

Activation of CD4⁺ T cell subsets is characterized by proliferation ofthe responsive T cell population coordinated with the selectionproduction of cytokines as described above dependent upon the type ofstimulation induced by an antigen presenting cell. The latter is definedas a highly specialized cell that can process antigens and display theirpeptide fragments on the cell surface together with molecules requiredfor lymphocyte activation. The specificity of CD4⁺ T cell activation isbased on the T cell antigen receptor (TCR) recognition of peptideantigens bound to MHC class II heterodimers on the surface of antigenpresenting cells (APC) The main APC for T cells are dendritic cells,macrophages and B cells. In addition, APC-derived non-antigeniccostimulatory signals play a contributory role in CD4⁺ T cellactivation.

The present invention utilizes a synthetic antigen presenting system,based on the natural immunological response mechanisms described above,to manipulate the activation of CD4⁺T cells by recombinant MHC class IImolecules in conjunction with one or more accessory molecules broadlyreferred to as costimulatory molecules. The latter include specificcostimulatory molecules, adhesion molecules and survival molecules, andthe like. In other aspects of the invention, the synthetic antigenpresenting system further contains antigen processing assistingmolecules that are useful in generating peptide-loaded MHC class IImolecules. In the context of this invention, the synthetic antigenpresenting systems are useful for activating CD4⁺ T cells in vitro andin vivo. These aspects are discussed in Section E on Methods of AlteringCD4⁺ T Cell Responses.

Thus, as mentioned above, the synthetic antigen presenting system of thepresent invention has at least two major components. The first componentis at least the extracellular portions of a recombinant MHC class IIheterodimer which is capable of binding to a peptide that provides thespecificity of CD4⁺ T cell activation via recognition by the TCR. Thesecond major component is at least the extracellular portion of at leastone accessory molecule that provides a non-antigenic specificcostimulatory signal in the activation of CD4⁺ T cells. In otherembodiments, the entire molecules of the antigen presenting system andthe non-antigen costimulatory signals are used.

For ease of description, MHC class II heterodimers will be discussedgenerally, with the understanding that an extracellular portion of theMHC molecule may be used in certain aspects of the invention. Theportion of the MHC molecule necessary for the present invention is thepart that binds to the antigenic peptide for presentation to the CD4⁺ Tcells.

The present invention allows the recombinant MHC class II heterodimersto be produced by vector-transformed synthetic antigen presenting cellswith the peptide already complexed with the MHC class II heterodimer.Alternatively, empty MHC class II heterodimers are produced that do notyet have a peptide complexed with them. This latter embodiment isparticularly useful as it allows for complexation with a particularpeptide or for screening of a library of peptides after the MHC class IIheterodimers are expressed.

1. MHC Class II Genes and Encoded Heterodimers

MHC class II molecules are cell surface glycoproteins that consist of anon-covalent complex of two chains, α and β, both of which span themembrane. A peptide-binding cleft is formed between the cooperatingchains. Peptides that bind MHC class II molecules are variable in lengthand have anchor residues that lie at various distances from the ends ofthe peptides thereby resulting in peptides having ends that are nottightly bound within the cleft of the binding pocket. See, Janeway andTravers, Immunobiology, Section 4-4, Current Biology LTD, 2nd ed., 1996.Further aspects of the presenting antigenic peptides are discussed inSection D.

In vivo, empty MHC class II heterodimers become destabilized and aresubsequently removed from the cell surface thereby preventing MHCmolecules for acquiring peptides from the surrounding extracellularfluid that would deleteriously effect T cell specificity. In presentinvention, the synthetic antigen presenting cell system allows for theproduction of empty MHC class II heterodimers on the cell surface thatare not subject to destabilizing events. As a consequence, loading of anantigenic peptide on a surface-expressed recombinant MHC class IIheterodimer is facilitated for subsequent use in manipulating CD4⁺activation and cytokine production.

The present invention, particular combinations of a selected MHC classII molecule with a coordinate antigen is facilitated by the presence ofconsensus nucleotide sequences in MHC class II genes. These regions,described further below, allow for the retrieval and use of the multipleMHC class II genes in the MHC complex in mammals as well as the multiplealleles of each gene. In other words, MHC is both polygenic andpolymorphic having respectively several genes and multiple alleles ofeach gene.

In humans, MHC is called HLA while in mouse it is referred to as H-2.Three pairs of MHC class II α- and β-chain genes in humans have beendesignated HLA-DP, HLA-DQ and HLA-DR. The HLA-DR cluster contains anextra β-chain gene. As such, the three sets of genes can give rise tofour types of MHC class II molecules. MHC class II genes and encoded α-and β-chains that are obtained from human genes are said to be of humanorigin. In mice, MHC class genes are designated H2-M, H2-A and H2-E. Aseach MHC class II molecule binds a different range of peptides, thepresence of multiple gene loci gives an individual the ability topresent a broad range of different peptides than if only one MHC classII molecules of each class were expressed at the cell surface.

While the polymorphic MHC class II genes encode corresponding proteinsthat vary by only one or a few amino acids, the different allelicvariants differ by up to 20 amino acids. As a result, MHC class IIdiversity expands the ability of antigen recognition by T cells.Moreover, via MHC restriction, T cells have been shown to recognizepeptide in the context of a particular MHC molecule but not whenpresented on another. Thus, T cell receptor specificity is imparted byboth peptide and by the MHC molecule binding it.

The MHC class II heterodimers of this invention containing selected α-and β-chains are obtained by amplification of MHC class II-encodinggenes and allelic variants thereof with specific pairs ofoligonucleotide primers. The nucleotide sequences of the primers allowfor amplification of the diversity of MHC class II genes and allelicvariants thereof based on the 5′ and 3′ consensus nucleotide sequencespresent in the genes within a category of genes. Specific nucleotidesequences of primer pairs for amplifying the α- and β-chains of humanHLA-DP, -DQ and -DR genes as well as those for amplifying murineIA^(d)-encoding heterodimers are presented in Example 2A.

The MHC class II-encoding genes are amplifiable from a variety ofcellular sources including B cells, macrophages and dendritic cells, allof which are present in the blood. The amplification conditions forobtaining amplified MHC class II-encoding genes with the primers of thisinvention are described in Section C.

The α- and β-chains comprising the MHC heterodimers of this inventionare useful in either anchored or soluble form. In the anchored form, therecombinant MHC heterodimer is anchored into the synthetic antigenpresenting cell from which it is expressed. Alternatively, a recombinantMHC heterodimer is anchored in a matrix comprising a support asdescribed in this invention after being secreted in soluble form. Thelatter is generated when a stop codon is engineered during theamplification procedure or thereafter into the nucleotide sequenceencoding the MHC class II α- and β-chains of choice preceding thetransmembrane domain.

2. Accessory Genes and Encoded Molecules

The accessory molecules of this invention, including costimulatorymolecules, adhesion molecules and survival molecules, are effective inconcert with the MHC class II heterodimer complexed with peptide inactivating CD4⁺ T cells to become armed effector T cells that recognizetarget cells. Naive T cells are activated to proliferate anddifferentiate into armed effector T cells when they encounter theirspecific antigen when presented by a peptide-loaded MHC class IIheterodimer on the surface of an APC. Activation not only requires therecognition of a foreign peptide fragment bound to a MHC class IIheterodimer but it also requires the simultaneous delivery of acostimulatory signal concurrently expressed by the APC.

Thus, the synthetic APC or matrices of this invention are characterizedby the presence not only of a particular MHC class II heterodimer butalso by the presence of one or more costimulatory molecules that arebroadly defined as accessory molecules. At least three types ofaccessory molecules, including specific costimulatory molecules,adhesion molecules, and survival molecules, are contemplated for use inpreparing synthetic APC or matrices of this invention

a. Costimulatory Molecules

A first type of an accessory molecule is composed of costimulatorymolecules such as B7.1 (previously known as B7 and also known as CD80)and B7.2 (also known as CD86) which binds to CD28 on T cells. B7.1 andB7.2 are structurally related glycoproteins that are homodimeric membersof the immunoglobulin superfamily. Other costimulatory molecules areanti-CD28 antibodies or the functional portions of such antibodies, e.g.Fab portions that bind to CD28. Ligation of CD28 by the above moleculeshas been shown to costimulate the growth of naive T cells. On activatedT cells, an additional receptor, CTLA-4, binds B7 molecules with ahigher affinity that that with CD28.

Recombinant B7 costimulatory molecules for use in the synthetic APC ormatrices of this invention are obtained by PCR as described for MHCclass II molecules. Preferred oligonucleotide primers and cellularsources for amplification therefrom as described in Example 2C.

b. Adhesion Molecules

Another major type of accessory molecule of the present invention is anadhesion molecule that also functions in T cell activation. Accessoryadhesion molecules include the various ICAM molecules, which includeintercellular adhesion molecule (ICAM) ICAM-1, ICAM-2, ICAM-3,lymphocyte function-associated antigen (LFA) LFA-1 and LFA-3. All ofthese molecules are members of the immunoglobulin superfamily. TheICAM-related members all bind to the T cell integrin, LFA-1. In additionto being expressed on APC including dendritic cells, macrophages and Bcells, ICAM-1 and ICAM-2 are also expressed on endothelium, therebymediating cell adhesion and subsequenct extravasation betweencirculating leukocytes and endothelium. ICAM-3, however, is onlyexpressed on leukocytes and is thought to play an important part inadhesion between T cells and APC.

The interaction between ICAM-1, -2 and -3 synergizes with a secondadhesive interaction between LFA-3 (CD58) and LFA-2 (CD2) that arerespectively expressed on an APC and a T cell surface.

Recombinant adhesion molecules for use in the synthetic APC or matricesof this invention are obtained by PCR as described for MHC class IImolecules. Preferred oligonucleotide primers and cellular sources foramplification therefrom as described in Example 2C.

c. Survival Molecules

A survival molecule is another type of an accessory molecule that playsa role in metabolic responses ranging from stimulatory to inducing celldeath. Thus, a survival molecule can also be referred to as a cell deathregulating molecule. A survival molecule is typically a protein but mayinclude other types of macromolecules such as carbohydrates, lipids andthe like. Survival molecules for use in the compositions and methods ofthis invention include Fas ligand, TNF-receptor, TNF, CD70, a Type IItransmembrane protein that is a member of the TNF family that binds toCD27, a member of the TNF receptor family. Fas ligand binds to thereceptor called Fas and receptor occupancy resulting in the induction ofapoptotic cell death of the cell expressing Fas receptor. CD27 isexpressed on resting T and B cells while CD70 is expressed on activatedT and B cells. Binding of CD70 to its receptor, CD27, induces T-cellcostimulation and the interaction may be important for the recruitmentof T cells from the unprimed T cell pool. Under other certainconditions, activation of the TNF receptor by TNF results in a similarresponse.

The recombinant survival molecules described above for use in thesynthetic APC or matrices of this invention are obtained by PCR asdescribed for MHC class II molecules. Preferred oligonucleotide primersand cellular sources for amplification therefrom as described in Example2C.

As shown in the Examples, particular combinations of a peptide bound toa recombinant MHC class II molecule used in conjunction with one or moreof the above-described recombinant accessory molecules activates T cellsinto armed effector T cells that are distinguishable into Th1inflammatory T cells and Th2 helper T cells.

3. Antigen Processing Assisting Genes and Encoded Molecules

a. HLA-DM

HLA-DM in humans and H2-M in mice is a MHC class II-like molecule thatis also encoded within the MHC class II gene clusters. HLA-DM, like MHCclass II, contains both α- and β-chain genes forming a heterodimer.However, unlike MHC class II molecules, peptide loading is not requiredfor stabilizing the molecule. HLA-DM facilitates the loading of peptidesonto newly formed MHC class II heterodimers following the removal of theinvariant chain as further described below. Recombinant HLA-DM iscontemplated for use in the compositions and methods of this inventionto assist in the loading of internally processed peptides.

b. Invariant Chain

The invariant chain is a specialized protein that binds to newly formedMHC class II heterodimers thereby forming a trimer with each subunit ofthe MHC class II heterodimer. The trimerized molecule prevents theloading of intracellular peptides present in the endoplasmic reticulumbut it also facilitates the export of the molecule from thatcompartment. Thereafter, the invariant chain is cleaved through multiplesteps resulting in a MHC class II heterodimer that can then be complexedwith processed peptides.

Thus, recombinant invariant chain is contemplated for use in thecompositions and methods of this invention to assist in the loading ofinternally processed peptides.

C. Nucleic Acids and Polynucleotides

1. PCR to Obtain Genes Encoding MHC Class II and Accessory Molecules

Nucleic acid sequences encoding MHC class II molecules, accessorymolecules and antigen processing assisting molecules of this inventionare obtained in a number of ways familiar to one of ordinary skill inthe art including direct synthesis, cloning, purification of DNA fromcells containing such genes, and the like. One expedient means to obtaingenes for encoding the molecules used in the compositions and methodsdescribed herein is by polymerase chain reaction (PCR) amplification onselected nucleic acid templates with selected oligonucleotide primerpairs as further described below.

Known, partial and putative human leukocyte antigen (HLA), the geneticdesignation for the human MHC, amino acid and nucleotide sequences,including the consensus sequence, are published (see, e.g., Zemmour andParham, Immunogenetics 33: 310-320 (1991)), and cell lines expressingHLA variants are known and generally available as well, many from theAmerican Type Culture Collection (“ATCC”). Therefore, using PCR, MHCclass II-encoding nucleotide sequences are readily operatively linked toan expression vector of this invention that is then used to transform anappropriate cell for expression therein.

Particularly preferred methods for producing the recombinant moleculesof the present invention rely on the use of preselected oligonucleotidesas primers in PCR to form PCR reaction products as described herein.

If a gene is to be obtained by PCR amplification, in general, twoprimers comprising a PCR primer pair, are used for each strand ofnucleic acid to be amplified. For the sake of simplicity, synthesis ofexemplary MHC class II heterodimer-encoding genes is discussed, but itis expressly to be understood that the PCR amplification methoddescribed is equally applicable to the synthesis of MHC class II allelicvariants, accessory molecules and antigen processing assistingmolecules, including those whose complete sequences are presentlyunknown.

In general, a first primer is referred to a a forward primer or a 5′primer as it has the same sequence as the top strand of template DNA andthus hybridizes to the bottom complementary strand.

A second primer is referred to as a backward primer or a 3′ primer as ithas the same sequence as the bottom strand and thus hybridizes to thecomplementary sequence on the top strand. Typically, in other words, oneprimer is complementary to the negative (−) or bottom strand of thenucleotide sequence and the other is complementary to the positive (+)or top strand.

In preferred aspects, both first and second primers are chosen tohybridize to (i.e., be complementary to) conserved regions within theMHC class II genes. However, primers can be designed to amplify specificMHC class II genes and allelic variants thereof by hybridizing to uniquerather than consensus sequences. For this aspect, the template sequenceis preferably known for design of such primer pairs.

One or both of the first and second primers can be designed to introduceinto the amplified product a nucleotide sequence defining anendonuclease recognition site. The site can be heterologous to the MHCclass II gene being amplified and typically appears at or near the 5′end of the primer. It may also be helpful to place a 4-base spacersequence proximal to the restriction site to improve the efficiency ofcutting amplification products with enzymes.

The primers of the invention for isolating specific nucleotide sequencesinclude oligonucleotides of sufficient length and appropriate sequenceso as to provide specific initiation of polymerization on a significantnumber of nucleic acids with the corresponding nucleotide sequence.Specifically, the term oligonucleotide primer as used herein refers to asequence comprising two or more deoxyribonucleotides or ribonucleotides,preferably more than three, and more preferably around 20, the sequenceof which is capable of initiating synthesis of a primer extensionproduct.

Experimental conditions conducive to synthesis include the presence ofnucleoside triphosphates and an agent for polymerization and extension,such as thermostable polymerases, and a suitable buffer, temperature andpH. The primer is preferably single stranded for maximum efficiency inamplification, but may be double stranded. If double stranded, theprimer is first treated to separate the two strands before being used toprepare extension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long andsubstantially complementary to prime the synthesis of extension productsin the presence of the inducing agent for polymerization and extensionof the nucleotides. The exact length of primer will depend on manyfactors, including temperature, buffer, and nucleotide composition. Theoligonucleotide primer typically contains 15-22 or more nucleotides,although it may contain fewer nucleotides. Alternatively, as is wellknown in the art, the mixture of nucleoside triphosphates can be biasedto influence the formation of mutations to obtain a library of mutatedrecombinant MHC class II-encoding molecules for use in presenting uniquepeptides to CD4⁺ T cells.

Oligonucleotide primers of the invention are employed in the PCRamplification process which is an enzymatic chain reaction that producesexponentially growing quantities of a nucleotide sequence. Annealing theprimers to denatured nucleic acids followed by extension with athermostable polymerase such as Thermophilus aquaticus (Taq) andPyrococcus furiosus (Pfu) (Hoffman La-Roche, Basal, Switzerland), andnucleotides, results in newly synthesized (+) and (−) strands. Becausethese newly synthesized sequences are also templates, repeated cycles ofdenaturing, primer annealing, and extension results in exponentialproduction of the DNA fragment defined by the primers. The product ofthe chain reaction is a discrete nucleic acid duplex with terminicorresponding to the ends of the specific primers employed. Those ofskill in the art will know of other amplification methodologies whichcan also be utilized to increase the copy number of target nucleicacids. These may include for example, ligation activated transcription(LAT), ligase chain reaction (LCR), and strand displacement activation(SDA), although PCR is the preferred method as described in the USPatents listed below.

The oligonucleotide primers of the invention may be prepared using anysuitable method, such as conventional phosphotriester and phosphodiestermethods as described above for synthesis of complementaryoligonucleotides or automated embodiments thereof. One method forsynthesizing oligonucleotides on a modified solid support is describedin U.S. Pat. No. 4,458,066.

Preferred primers for amplifying MHC class II genes, accessory moleculegenes, and antigen processing assisting molecule genes are described inExample 2.

PCR amplification methods are described in detail in U.S. Pat. Nos.4,683,192, 4,683,202, 4,800,159, 4,965,188 and 5,395,750, thedisclosures of which are hereby incorporated by reference, and at leastin several texts including “PCR Technology: Principles and Applicationsfor DNA Amplification”, H. Erlich, ed., Stockton Press, New York (1989);and “PCR Protocols: A Guide to Methods and Applications”, Innis et al.,eds., Academic Press, San Diego, Calif. (1990). Various preferredmethods and primers used herein are described hereinafter and are alsodescribed by Zemmour, et al., Immunogenetics, 33:310-20 (1991), byAusebel, et al., In Current Protocols in Molecular Biology, Wiley andSons, New York (1993) and by Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, (1989). Particular PCRmethods including nested PCR, overlap PCR, reverse-transcriptase-PCR,and the like that are well known to one of ordinary skill in the art arecontemplated for use in obtaining the recombinant molecules of thisinvention.

In alternative embodiments, the PCR process is used not only to producea variety of MHC class II-encoding molecules, but also to inducemutations which may emulate those observed in the highly-polymorphic MHCloci, or to create diversity from a single parental clone and therebyprovide a MHC class II-encoding DNA “library” having a greaterheterogeneity.

2. Expression Vectors

The present invention contemplates plasmid expression vectors insubstantially pure form capable of directing expression of MHC classII-encoding genes, accessory molecule genes and antigen processingassisting genes to produce the corresponding recombinant proteins. Forsimplicity, the above genes are herein referred to collectively aspolypeptide-encoding nucleotide sequences. Vectors capable of directingthe expression of genes to which they are operatively linked arereferred to herein as “expression vectors” or “expression plasmids”,both of which are also referred to as “plasmids”.

As used herein, the term “vector” or “plasmid” refers to a nucleic acidmolecule capable of transporting between different genetic environmentsanother nucleic acid to which it has been operatively linked. Preferredvectors are those capable of autonomous replication and expression ofstructural gene products present in the DNA segments to which they areoperatively linked. Vectors, therefore, preferably contain the repliconsand selectable markers that are compatible with the host selectionsystem. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication.

A plasmid of this invention is a circular double-stranded plasmid thatcontains at least a regulation region having elements capable ofactivating transcription of the translatable polypeptide-encodingnucleotide sequences of this invention. The plasmid further contains atranslatable nucleotide sequence from which the desired encodedpolypeptides of this invention are expressed. Thus, the vectors are saidto be capable of directing the expression of the recombinantpolypeptides described herein as encoded from the correspondingexpressible genes.

A preferred vector for use according to the present invention is aplasmid; more preferably, it is a high copy number plasmid. It is alsopreferable that the vector of choice be best suited for expression inthe chosen host.

Such expression vectors contain a promotor sequence in the regulatoryregion which facilitates the efficient transcription of an insertedgenetic sequence in the host. Preferably, the vector contain aninducible promoter sequence, as inducible promoters tend to limitselection pressure against cells into which such vectors (which areoften constructed to carry non-native or chimeric nucleotide sequences)have been introduced. The expression vector also typically contains anorigin of replication as well as specific genes which allow phenotypicselection of the transformed cells. The DNA segment can be present inthe vector operatively (also referred to as operably) linked toregulatory elements, for example, a promoter (e.g., T7, metallothioneinI, or polyhedrin promoters).

In a separate embodiment, a plasmid also contains a gene, the expressionof which confers a selective advantage, such as a drug resistance, to ahost cell when introduced or transformed into that cell. Typicalprokaryotic and eukaryotic drug resistance genes respectively conferresistance to ampicillin or tetracyclin and to neomycin (G418 orGeneticin). Other drug resistance markers include chloramphenicol,kanamycin, streptomycin, carbenicillin, mercury, rifampcin, rifampicin,fusaric acid, and the like.

The choice of vector to which the regulatory region and nucleotidesequences for encoding polypeptides of the present invention isoperatively linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., replication or protein expression,and the host cell to be transformed, these being limitations inherit inthe art of constructing recombinant DNA molecules.

Operatively linking refers to the covalent joining of nucleotidesequences, preferably by conventional phosphodiester bonds, into onestrand of DNA, whether in single or double stranded form. Moreover, thejoining of nucleotide sequences results in the joining of functionalelements such as response elements in regulatory regions with promotersand downstream polypeptide-encoding nucleotide sequences as describedherein.

One typical method for operatively linking inserts into expressionplasmids is by directional ligation. This is accomplished through asequence of nucleotides that are adapted for directional ligation. Sucha sequence is referred to commonly as a polylinker that is a region ofthe DNA expression vector that (1) operatively links for replication andtransport the upstream and downstream translatable DNA sequences and (2)provides a site or means for directional ligation of a DNA sequence intothe vector. Typically, a directional polylinker is a sequence ofnucleotides that defines two or more restriction endonucleaserecognition sequences, or restriction sites. Upon restriction cleavage,the two sites yield cohesive termini to which a translatable DNAsequence can be ligated to the DNA expression vector. Preferably, thetwo restriction sites provide, upon restriction cleavage, cohesivetermini that are non-complementary and thereby permit directionalinsertion of a translatable DNA sequence into the cassette.

A variety of host-expression vector systems may be utilized to express apolypeptide encoded by a nucleotide sequence. These include but are notlimited to microorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining a polypeptide-encoding nucleotide sequence; yeast transformedwith recombinant yeast expression vectors containing apolypeptide-encoding nucleotide sequence; plant cell systems infectedwith recombinant virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinantplasmid expression vectors (e.g., Ti plasmid) containing apolypeptide-encoding nucleotide sequence; insect cell systems infectedwith recombinant virus expression vectors (e.g., baculovirus) containinga polypeptide-encoding nucleotide sequence; or animal cell systemsinfected with recombinant virus expression vectors (e.g., retroviruses,adenovirus, vaccinia virus) containing a polypeptide-encoding nucleotidesequence, or transformed animal cell systems engineered for stableexpression. In such cases where glycosylation may be important,expression systems that provide for translational and post-translationalmodifications may be used; e.g., mammalian, insect, yeast or plantexpression systems.

Any of these systems are useful in practicing the methods of thisinvention. With any of the above expression systems, the selected hostis then used for expression of at least one MHC class II heterodimeralone or in conjunction with at least one accessory molecule furtherwith or without an antigen processing accessory molecule depending onthe actual mechanism for loading peptides, i.e., internally orexternally.

Depending on the host/vector system utilized, any of a number ofsuitable transcription and translation elements, including constitutiveand inducible promoters, transcription enhancer elements, transcriptionterminators, etc. may be used in the expression vector (see e.g.,Bitter, et al., Methods in Enzymology, 153:516-544, (1987)). Forexample, when cloning in bacterial systems, inducible promoters such asPl of bacteriophage λ, Plac, Ptrp, Ptac (Ptrp-lac hybrid promoter) andthe like may be used. When cloning in mammalian cell systems, promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the retrovirus long terminalrepeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter)may be used. Promoters produced by recombinant DNA or synthetictechniques may also be used to provide for transcription of apolypeptide-encoding nucleotide sequence.

In bacterial systems a number of expression vectors may beadvantageously selected for expression of a polypeptide-encodingnucleotide sequence according to the methods of this invention. Forexample, when large quantities are to be produced, vectors which directthe expression of high levels of fusion protein products that arereadily purified may be desirable. Those which are engineered to containa cleavage site to aid in recovering the protein are preferred. Suchvectors include but are not limited to the E. coli expression vectorpUR278 (Ruther, et al., EMBO J., 2:1791, (1983)), in which thepolypeptide-encoding nucleotide sequence may be ligated into the vectorin frame with the LacZ coding region so that a hybrid polypeptide-LacZprotein i-s produced; pIN vectors (Inouye & Inouye, Nuc. Acids Res.,13:3101-3109, (1985); Van Heeke & Schuster, J. Biol. Chem.,264:5503-5509, (1989)); and the like.

In one embodiment, the vector utilized includes prokaryotic sequencesthat facilitate the propagation of the vector in bacteria, i.e., a DNAsequence having the ability to direct autonomous replication andmaintenance of the recombinant DNA molecule extra-chromosomally whenintroduced into a bacterial host cell. Such replicons are well known inthe art.

Those vectors that include a prokaryotic replicon also typically includeconvenient restriction sites for insertion of a recombinant DNA moleculeof the present invention. Typical of such vector plasmids are pUC8,pUC9, pBR322, and pBR329 available from BioRad Laboratories, (Richmond,Calif.) and pPL available from Pharmacia, (Piscataway, N.J.), andpBLUESCRIPT and pBS available from Stratagene, (La Jolla, Calif.). Avector of the present invention may also be a Lambda phage vectorincluding those Lambda vectors described in Molecular Cloning: ALaboratory Manual, Second Edition, Maniatis et al., eds., Cold SpringHarbor, N.Y. (1989).

In another preferred embodiment, plasmid vectors for use in the presentinvention are also compatible with eukaryotic cells. Eukaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors provide convenientrestriction sites for insertion of the desired recombinant DNA molecule,and further contain promoters for expression of the encoded genes whichare capable of expression in the eukaryotic cell, as discussed earlier.Typical of such vectors are pSVO and pKSV-10 (Pharmacia), andpPVV-1/PML2d (International Biotechnology, Inc.), and pTDT1 (ATCC, No.31255).

In addition, in eukaryotic plasmids, one or more transcription units arepresent that are expressed only in eukaryotic cells. The eukaryotictranscription unit consists of noncoding sequences and sequencesencoding selectable markers. The expression vectors of this inventionalso contain distinct sequence elements that are required for accurateand efficient polyadenylation. In addition, splicing signals forgenerating mature mRNA are included in the vector. The eukaryoticplasmid expression vectors can contain viral replicons, the presence ofwhich provides for the increase in the level of expression of clonedgenes. A preferred replication sequence is provided by the simian virus40 or SV40 papovavirus.

A preferred expression system for producing the recombinant moleculesfor use in this invention is an insect system. In one such system,Autographa californica nuclear polyhedrosis virus (AcNPV) is used as avector to express foreign genes. The virus grows in Spodopterafrugiperda (Sf9) cells. The polypeptide-encoding nucleotide sequences ofthis invention may be cloned into non-essential regions (in Spodopterafrugiperda for example the polyhedrin gene) of the virus and placedunder control of an AcNPV promoter (for example the polyhedrinpromoter). Successful insertion of the polypeptide-encoding nucleotidesequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect cells in which the inserted gene isexpressed. See Smith, et al., J. Biol. Chem., 46:584, (1983); Smith,U.S. Pat. No. 4,215,051.

In preferred embodiments, the host cell population is a Drosophila cellculture that requires a compatible vector including vectors functionallyequivalent to those such as p25-lacZ (see Bello and Couble, Nature,346:480 (1990)) or pRmHa-1, -2, or -3 (see Bunch, et al., Nucl. AcidsRes., 16:1043-1061 (1988)). In the preferred embodiment, the vector ispRmHa-3, which is shown in FIG. 1C. This vector includes ametallothionein promoter, which is preferably upstream of the site atwhich the MHC sequence is inserted, and the polyadenylation site ispreferably downstream of said MHC sequence. Insect cells and, inparticular, Drosophila cells are preferred hosts according to thepresent invention. Drosophila cells such as Schneider-2 (S2) cells, asfurther described in Section D, have the necessary trans-acting factorsrequired for the activation of the promoter and are thus even morepreferred.

The expression vector pRmHa-3 is based on the bacterial plasmid pRmHa-1(FIG. 1A), the latter of which is based on plasmid pUC18 and isdeposited with the American Type Culture Collection (ATCC, Rockville,Md.), having the accession number 37253. The pRmHa-3 vector contains thepromoter, the 5′ untranslated leader sequence of the metallothioneingene with the Eco RI and Stu I sites removed as shown in FIG. 1C. Italso contains the 3′ portion of the Drosophila ADH gene including thepolyadenylation site. Therefore, cloned DNA is transcriptionallyregulated by the metallothionein promoter and polyadenylated.Construction of the pRmHa-1 plasmid is described in Bunch, et al., Nucl.Acids Res. 16: 1043-1061 (1988). Construction of the pRmHa-3 and pRmHa-2plasmids (the latter of which has a metallothionein promoter sequencethat may be removed as an Eco RI fragment) is described in the Examples.With regard to pRmHa-3, a preferred plasmid for use according to thepresent invention, Pst I, Sph I and Hind III are in the promoterfragment and therefore are not unique. Xba I is in the ADH fragment (4bases from its 3′ end) and is also not unique. The following restrictionsites are, however, unique in pRmHa-3 to facilitate cloning of therecombinant genes of this invention: Eco RI, Sac I, Kpn I, Sma I, BamHI, Sal I, Hinc 2, and Acc I.

Mammalian cell systems that utilize recombinant viruses or viralelements to direct expression may be engineered. For example, when usingadenovirus expression vectors, the coding sequence of a polypeptide maybe ligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted into the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing the polypeptide in infected hosts(e.g., see Logan & Shenk, Proc. Natl. Adad. Sci., USA, 81:3655-3659,(1984)). Alternatively, the vaccinia virus 7.5K promoter may be used.(e.g., see, Mackett, et al., Proc. Natl. Acad. Sci. USA, 79:7415-7419,(1982); Mackett, et al., J. Virol., 49:857-864, (1984); Panicali, etal., Proc. Natl. Acad. Sci. USA, 79:4927-4931, (1982)). Of particularinterest are vectors based on bovine papilloma virus which have theability to replicate as extrachromosomal elements (Sarver, et al., Mol.Cell. Biol., 1:486, (1981)). Shortly after entry of this DNA into mousecells, the plasmid replicates to about 100 to 200 copies per cell.Transcription of the inserted cDNA does not require integration of theplasmid into the host's chromosome, thereby yielding a high level ofexpression. These vectors can be used for stable expression by includinga selectable marker in the plasmid, such as the neo gene. Alternatively,the retroviral genome can be modified for use as a vector capable ofintroducing and directing the expression of a polypeptide-encodingnucleotide sequence of this invention in host cells (Cone & Mulligan,Proc. Natl. Acad. Sci., USA, 81:6349-6353, (1984)). High levelexpression may also be achieved using inducible promoters, including,but not limited to, the metallothionine IIA promoter and heat shockpromoters.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. Rather than using expression vectors whichcontain viral origins of replication, host cells can be transformed witha cDNA controlled by appropriate expression control elements (e.g.,promoter and enhancer sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. As mentionedabove, the selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines.

For example, following the introduction of foreign DNA, engineered cellsmay be allowed to grow for 1-2 days in an enriched media, and then areswitched to a selective media. A number of selection systems may beused, including but not limited to the herpes simplex virus thymidinekinase (Wigler, et al., Cell, 11:223, (1977)), hypoxanthine-guaninephosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci.USA, 48:2026, (1962)), and adenine phosphoribosyltransferase (Lowy, etal., Cell, 22:817, (1980)) genes, which can be employed in tk⁻, hgprt⁻or aprt⁻ cells respectively. Also, antimetabolite resistance-conferringgenes can be used as the basis of selection; for example, the genes fordhfr, which confers resistance to methotrexate (Wigler, et al., Proc.Natl. Acad. Sci. USA, 77:3567, (1980); O'Hare, et al., Proc. Natl. Acad.Sci. USA, 78:1527, (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072,(1981)); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., J. Mol. Biol., 150:1, (1981)); and hygro,which confers resistance to hygromycin (Santerre, et al., Gene, 30:147,(1984)). Recently, additional selectable genes have been described,namely trpB, which allows cells to utilize indole in place oftryptophan; hisD, which allows cells to utilize histinol in place ofhistidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. USA, 85:804,(1988)); and ODC (ornithine decarboxylase) which confers resistance tothe ornithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine,DFMO (McConlogue L., In: Current Communications in Molecular Biology,Cold Spring Harbor Laboratory ed., (1987).

Both prokaryotic and eukaryotic expression vectors are familiar to oneof ordinary skill in the art of vector construction and are described byAusebel, et al., In Current Protocols in Molecular Biology, Wiley andSons, New York (1993) and by Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, (1989).

For producing recombinant MHC class II α- and β-chains, accessorymolecules and antigen processing assisting molecules for use in thecompositions and methods of this invention, the respective nucleotideregions are operatively inserted into an expression vector of thisinvention as described herein. As described in Section B, nucleic acidsencoding the recombinant polypeptides of this invention are obtained ina number of ways one of which is by PCR amplification. One of thenucleotide segments to be operatively linked to vector sequences encodesat least a portion of MHC class II α- and β-chains. Preferably, therespective nucleotide sequences for encoding the complete α- andβ-chains are separately inserted into an expression vector forexpression therefrom; however, it is also feasible to construct a vectorwhich also includes some non-coding MHC sequences as well. The sequencesfor encoding accessory molecules and antigen processing assistingmolecules are similarly inserted into separate expression vectors.

Alternatively, the invention contemplates the presence of more than onepolypeptide-encoding gene being present within the same vector, theexpression of which is driven by separate regulatory elements, such as apromoter. In other words, the nucleic acids for encoding both α- andβ-chains may be operatively ligated to the same expression vector withor without one or more nucleic acid sequences encoding accessorymolecules. Thus, all possible combinations of expression vectorconstruction for producing the recombinant proteins of this inventionare contemplated.

In addition to the complete encoding nucleotide sequences as describedabove, soluble forms of the expressed recombinant polypeptides of thisinvention are contemplated. The soluble form differs from thenon-soluble form in that it contains a “stop” codon inserted prior tothe transmembrane domain or other functional location to generatesoluble non-membrane anchorable proteins.

D. Synthetic Antigen Presenting Cells and Matrices for PeptidePresentation

1. Synthetic Antigen Presenting Cells and Matrices

In accordance with the present invention, the recombinant MHC class IIheterodimers and at least one accessory molecule are operably linked toa matix comprising a support such that the MHC class II and accessorymolecules are present in sufficient numbers to activate a population ofCD4⁺ T cells lymphocytes when presented with a peptide complexed to theextracellular portion of the MHC molecule. The peptide can be bound tothe MHC class II heterodimer before or after it is linked to thesupport.

The support can take on many different forms. It can be a solid supportsuch as a plastic or metal material, it can be a porous material such ascommonly used in separation columns, it can be a liposome or red bloodcell, or it can even be a cell or cell fragment. As discussed in moredetail below, in the case where a cell serves as a support, the MHCclass II and accessory molecules can be produced by the cell forpresentation on that cell or for presentation on another support thatcan include a separate cell.

In the former situation, the MHC molecule is then linked to the cell byat least the transmembrane domain if not also the cytoplasmic domainwhich would not be present in a soluble form of MHC class II. In thelatter situation, the extracellular portions of MHC class II moleculeand accessory molecule can be linked to a support by providing anepitope which reacts to an antibody immobilized on the support. Inaddition, the MHC or assisting molecules can be produced with or linkedto (His)₆ which would react with nickel in forming part of the support.Other means to immobilize or link MHC molecules to a support are wellknown in the art.

As discussed above, the support can be a cell membrane or an entirecell. In such a case, an eukaryotic cell line is modified to become asynthetic antigen presenting cell line for use in presenting peptide inthe context of MHC class II to T cell lymphocytes. Because empty MHCmolecules are thermolabile, it is preferred that the cell culture bepoikilotherm and various cell lines are discussed in detail below.

A preferred cell line of the present invention is capable of continuousgrowth in culture and capable of expressing mammalian MHC class IImolecules and accessory molecules on the cell surface. Any of a varietyof transformed and non-transformed cells or cell lines are appropriatefor this purpose, including bacterial, yeast, insect, and mammalian celllines. (See, e.g., Current Protocols in Molecular Biology, John Wiley &Sons, NY (1991), for summaries and procedures for culturing and using avariety of cell lines, e.g., E. coli and Sarcomyces cerevisiae).

Preferably, the cell line is a eukaryotic cell line. More preferably,the cell line is poikilothermic (i.e., less sensitive to temperaturechallenge than mammalian cell lines). More preferably, it is an insectcell line. Various insect cell lines are available for use according tothe present invention, including moth (ATCC CCL 80), armyworm (ATCC CRL1711), mosquito larvae (ATCC lines CCL 125, CCL 126, CRL 1660, CRL 1591,CRL 6585, CRL 6586), silkworm (ATCC CRL 8851) and butterfly (Spodopterafrugiperda (Sf9 cells, ATCC CRL 1711). In a preferred embodiment, thecell line is a Drosophila cell line such as a Schneider cell line (seeSchneider, J. Embryol. Exp. Morph., 27:353-365 (1972)); preferably, thecell line is a Schneider 2 (S2) cell line (S2/M3) adapted for growth inM3 medium (see Lindquist, et al., Drosophila Information Service, 58:163(1982)). Schneider 2 (S2) cells have been deposited pursuant to BudapestTreaty requirements with the American Type Culture Collection (ATCC),Rockville, Md., on Feb. 18, 1992, and was assigned accession number CRL10974.

To generate a synthetic antigen presenting cell of this invention, oneor more expression vectors for directing the expression of a selectedMHC class II heterodimer in conjunction with one or more accessorymolecules is introduced into a recipient host cell. In addition; inalternative embodiments, vectors for expressing antigen processingassisting molecules including HLA-DM and invariant chain are alsointroduced into the recipient cells. The genes for the above have beendescribed in Sections B and C.

Thus, in order to prepare synthetic antigen presenting cells ormatrices, the expression vectors for encoding recombinant polypeptidesof this invention are transfected, i.e, introduced, into a selected hostcell. The selection of expression vectors as well as the constructionthereof is dependent upon the desired outcome for CD4⁺ activation aspreviously discussed as as reiterated below. Transfection, also referredto as transformation, may be accomplished via numerous methods,including the calcium phosphate method, the DEAE-dextran method, thestable transfer method, electroporation, or via the liposome mediationmethod. Numerous texts are available which set forth known transfectionmethods and other procedures for introducing nucleotides into cells;see, e.g., Current Protocols in Molecular Biology, John Wiley & Sons, NY(1991). Following introduction of one or more vectors, the recipientcell is said to be transformed, the selection of which can either betransient or stable.

A culture of cells is first established. A cell line is chosen fortransfection because it lacks at least one of the genes beingintroduced. It has been found that insect cells are advantageous notonly because they are poikilothermic, but because they lack these genesand the mechanisms which would otherwise produce MHC molecules bound topeptides. This allows for greater control over the production ofpeptide-bound MHC molecules, and the production of empty MHC molecules.

The selected cells are then transformed by the introduction of anexpression vector containing an expressible MHC class II α-chain geneoperably linked to a first promoter and an expressible MHC class IIβ-chain gene operably linked to a secong promoter. A first expressibleaccessory molecule gene operably linked to a third promoter in a vectoris also introduced into the above cell. In a further embodiment, anexpressible antigen processing assisting gene operably linked to afourth promoter in a vector is introduced into the above cell.

In a more preferred embodiment, the vector comprises Drosophilaexpression plasmid pRmHa-3, described in Section C, into whichexpressible nucleotide sequences encoding the above recombinant proteinshave been inserted using techniques disclosed herein. Preferably, thenucleotide sequences encoding the MHC class II chains, those encoding atleast one accessory molecule and those encoding antigen processingassisting molecules are operably linked to separate expression plasmidsthat are individually cotransfected into the cultured cells.Alternatively, the nucleotide sequences may be operably linked toseparate promoters in the same expression plasmid and cotransfected viathat same plasmid. The MHC class II α- and β-chains are preferably froma different species, more preferably, a homeotherm such as mammals and,optimally, humans.

It is preferred that at least one of the genes and, in particular, theMHC class II chain genes be linked to an inducible promoter. This allowscontrol over the production of MHC molecules so that they are onlyproduced at a time when the peptide of interest is available eitherinternally or externally and presented in the culture to react with theproduced MHC molecules. This minimizes undesirable MHC molecule/peptidecomplexes.

Thus, the preferred cell line is a poikilotherm cell line that hasseparate vectors each containing a MHC class II α- and β-chain generespectively operably linked to a first and second promoter. Preferably,the promoters are inducible to control the expression of the MHC classII chains. In addition, the cell contains a third vector containing atleast a first expessible accessory molecule gene operably lined to athird promoter. In a further embodiment, the cell also contains a fourthvector containing an expressible antigen processing assisting geneoperably linked to a fourth promoter. It is preferred that the cellassembles empty MHC molecules and presents them on the cell surface sothat the peptides specific for a particular MHC class II haplotype orfor a variant allele can be selected as desired.

The selection of compatible MHC class II α- and β-chain genes for use inconjunction with one or more particular accessory molecule-encodinggenes is dependent upon the T cell activation profile desired. Forexample, as described in the Examples, recombinant B7.1 or B7.2 alone ortogether in conjunction with recombinant murine IA^(d) MHC class IIexpressed on the surface of Drosophila APC resulted in proliferation ofCD4⁺ T cells having a Th2 profile of increased production of IL-4 andIL-10. In contrast, when either B7.1 or B7.2 were expressed on thesurface of Drosophila APC with ICAM-1 along with the same MHC molecules,the activation of CD4⁺ T cells resulted in a Th1 profile with increasedIL-2 production and decreased IL-4 and IL-10 production.

Thus, the invention contemplates the production of synthetic APC havingon the cell surface any combination of a MHC class II haplotypeheterodimer with any one of the accessory molecules of this invention.Particularly preferred combinations include MHC class II with either acostimulatory molecule including B7.1 or B7.2, an adhesion moleculeincluding ICAM-1, ICAM-2, ICAM-3 or LFA-3, or a survival moleculeincluding Fas ligand (FasL). As described above, more than one of eachcategory of accessory molecules can be co-expressed, as for example,B7.1 and B7.2. Alternative preferred embodiments include thepermutations where two accessory molecules of different categories areco-expressed on the cell surface. In other words, an adhesion moleculewith a costimulatory molecule, and adhesion molecule with a survivalmolecule, a costimulatory molecule with a survivial molecule. In afurther embodiment, three accessory molecules of the differentcategories described herein are co-expressed on the APC surface. It isalso contemplated that in all of these embodiments, more than one memberof a particular category may be expressed in conjunction with more thanone member of other categories. The particular selected combinations areeffective on the surface of the cells from which they are expressed orwhen anchored on the surface of a matrix of this invention. The actualcombinations prepared are selected on the basis of T cell activationoutcome in view of the expressed MHC class II molecules complexed withantigenic peptide. Thus, depending on the complex of MHC classII/peptide, the T cell activation outcome may be distinct despite havingthe same expressed accessory molecules.

In a further embodiment, antigen processing assisting genes areco-transfected with any of the above-described combinations to providefor enhanced internal peptide processing and loading. This aspect thusdoes not involve the generation of empty MHC class II molecules on thecell surface for subsequent peptide complexation. Rather, the expressionof invariant chain, HLA-DM or lysosomal enzymes is utilized to allow foroptimal processing and loading of proteolytic peptide fragmentsfollowing cell internalization. The APC of this invention thus canfunction in either motif of having the recombinant MHC class IIheterodimers being loaded either intracellularly or extracellularly.

Successfully transformed cells, i.e., cells that contain at least oneexpression vector capable of directing the expression of nucleotidesequences according to the present invention, can be identified viawell-known techniques. For example, cells resulting from theintroduction of a cDNA or rDNA of the present invention can be cloned toproduce individual colonies. Cells from those colonies can be harvested,lysed, and their DNA content examined for the presence of the rDNA usinga method such as that described by Southern, J. Mol. Biol. 98: 503(1975). In addition to directly assaying for the presence of rDNA,successful transformation or transfection may be confirmed by well-knownimmunological methods when the rDNA is capable of directing theexpression of a subject MHC class II protein of accessory molecule. Forexample, cells successfully transformed with one or more expressionvectors may produce proteins displaying particular antigenic propertieswhich are easily determined using the appropriate antibodies, such asanti-class II of particular haplotypes. In addition, successfultransformation/transfection may be ascertained via the use of anadditional vector bearing a marker sequence, such as neomycinresistance, as described hereinabove.

It is also preferable that the culture be stable and capable ofsustained growth at reduced temperatures. For example, it is preferredthat the culture be maintained at about room temperature, e.g., about24-27° C. In other embodiments, the culture is maintained at highertemperatures, particularly during the process of activating CD4⁺ cells.It is thus preferred that a culture according to the present inventionbe capable of withstanding a temperature challenge of about 30° C. toabout 37° C.

In order to prepare the culture for expression of empty or MHC class IImolecules in conjunction with a least one accessory molecule of thisinvention and optionally antigen processing assisting molecules, theculture may first require stimulation, e.g., via CuSO₄ induction, for apredetermined period of time. After a suitable induction period, e.g.,about 12-48 hours, peptides may be added at a predeterminedconcentration (e.g., about 0.2 μg/ml to 20 μg/ml). Proteins and peptidesfor both internal and external loading are prepared as discussed below.After a further incubation period, e.g., for about 12 hours at 27° C.,the culture is ready for use in the activation of CD4⁺ cells. While thisadditional incubation period may be shortened or perhaps omitted, theculture tends to become increasingly stable to temperature challenge ifit is allowed to incubate for a time prior to addition of resting ornaive CD4⁺ cells. For example, cultures according to the presentinvention to which peptide has been added are capable of expressingsignificant amounts of peptide-loaded MHC class II molecules even whenincubated for extended periods of time at 37° C.

Nutrient media useful in the culturing of transformed host cells arewell known in the art and can be obtained from numerous commercialsources. In embodiments wherein the host cell is mammalian, a“serum-free” medium is preferably used.

The resulting recombinant expressed MHC class II molecules bind to aparticular peptide and are present in sufficient numbers with at leastone accessory molecule on the surface of the APC to activate apopulation of T cell lymphocytes against the MHC class II/peptidecomplex.

Where the cell line already produces one or more of the desiredmolecules, it is only necessary to transfect the culture with anexpressible gene for the gene which is lacking in the cells. Forexample, if the cells already present the MHC molecules on theirsurface, it is only necessary to transfect the culture with a vectorcontaining an expressible gene for the accessory molecule.

As previously discussed, a protein or peptide can be introduced into thecell culture at the time the cells are producing MHC class II moleculesfor internal processing. Through methods such as osmotic shock, thepeptides can be introduced in the cell and bind to the produced MHCmolecules. Alternatively, particularly in the case poikilotherm celllines, the MHC molecules will be presented empty on the cell surface.The peptide can then be added to the culture and bound to the MHCmolecules as desired. For simplicity, while one peptide is describedherein, the methods of this invention contemplate the screening ofpeptide libraries for identifying novel antigenic peptides for use inthe therapeutic methods of this invention.

After the cells are produced having a MHC class II heterodimer and atleast one accessory molecules on the cell surface, the cells can belyophilized to generate cell fragments for use in activating apopulation of CD4⁺ T cell lymphocytes.

Transfected cultures of cells are also used to produce extracellularportions of MHC class II molecules and accessory molecules. The use ofextracellular portions in conjunction with supports such as solidsupports has certain advantages of production. Where living cells areused to provide a synthetic antigen presenting cell, at least threegenes, two to produce the MHC class II heterodimer and one for theaccessory molecule must be introduced to the cell. Often, additionalgenes such as for antibiotic resistance are also transfected.

Where a solid support system is being used, one cell line is used toproduce the extracellular portions of MHC class II molecules whileanother cell line is used to produce the extracellular portion of anaccessory molecule. The MHC molecule portions and the accessory moleculeportions are then harvested from their respective cultures. Themolecules are then linked to an appropriate support in sufficientnumbers to activate a population of T cells. From a productionstandpoint, two different cultures can be used, but it is also possibleto use the same culture, however, requiring that the culture betransfected with the additional gene for expressing the extracellularportion of an accessory molecule.

A further modification of this embodiment is to provide a third cultureof cells which is transfected with an expressible second accessorymolecule gene. For example, the second culture of cells producesextracellular portions of the costimulatory molecule while the thirdculture of cells produce an extracellular portion of an adhesionmolecule. The adhesion molecule portions are harvested and linked to thesupport. In preparing the extracellular portions of a MHC class IIheterodimer to be linked to a support, soluble molecules are prepared aspreviously discussed. These molecules generally lack the transmembraneand cytoplasmic domain in the MHC molecule.

2. Peptides

Virtually all cellular proteins in addition to viral antigens arecapable of being used to generate relevant peptide fragments that serveas potential MHC class II-specific peptides. The methods andcompositions of this invention provide for MHC class II molecules thathave an increased capacity to specifically activate CD4⁺ cells.

The peptides of the present invention bind to MHC class II molecules.The binding occurs under biological conditions which can be created invivo as well as in vitro. The exact nature of the binding of thepeptides need not be known for practice of the invention.

Peptides that bind MHC class II molecules are variable in length andtheir anchor residues lie at various distances from the ends of thepeptide. In one aspect, the peptides prepared for loading onto the MHCmolecules are of a single species; i.e., that all peptides loaded ontothe MHC be identical in size and sequence so as to produce monoantigenicpeptide-loaded MHC class II molecules. In alternative embodiments,peptides are heterogenous and may comprise a random library of peptidesto allow for selection of unique members that result in desired T cellactivation profiles as previously described. The production andscreening of random synthetic peptide libraries is familiar to one ofordinary skill in the art and is described in U.S. Pat. Nos. 5,556,762,5,510,240, 5,498,530, 5,432,018, 5,382,513, 5,338,665 and 5,270,170, thedisclosures of which are hereby incorporated by reference.

Peptides may be presented to the cells via various means. Preferably,peptides are presented in a manner which allows them to enter anintracellular pool of peptides. For example, peptides may be presentedvia osmotic loading. Typically, peptides are added to the culturemedium. The peptides may be added to the culture in the form of anintact polypeptide or protein which is subsequently degraded viacellular processes, e.g., via enzymatic degradation. Alternatively, theintact polypeptide or protein may be degraded via some other means suchas chemical digestion (e.g. cyanogen bromide) or proteases (e.g.chymotrypsin) prior to its addition to the cell culture. In otherembodiments, the peptides are presented in smaller segments which may ormay not comprise antigenic amino acid sequences in conjunction with aparticular MHC class II haplotype.

Preferably, a sufficient amount of protein(s) or peptide(s) is added tothe cell culture or synthetic matrix to allow the MHC class II moleculesto bind and subsequently present a large density of the peptide.preferably, with the same kind of peptide attached to each MHCheterodimer, on the surface of the synthetic APC or matrices of thisinvention.

In another embodiment of the invention, peptides are added totransfected cells of the present invention in order to enhance thethermostability of the MHC molecules expressed by the cells. As notedabove, peptides are preferably added to the culture medium. Antigenicpeptides that bind to the MHC class II molecules serve tothermostabilize the MHC molecules and also increase the cell surfaceexpression. Cultures with added peptides which bind to the MHC moleculesare thus significantly less susceptible to temperature challenge thancultures without added peptide.

E. Methods of Altering CD4⁺ T Cell Responses

1. Th1 and Th2 CD4⁺ T Cell-Mediated Diseases

Inducing a naive T cell into a desired activated T cell type ordeviating the effector function of an activated T cell from a Th1 typeto a Th2 type and visa versa is one of the aims of the presentinvention, especially with regard to therapeutic methods in treatingvarious CD4⁺ T cell-mediated disease conditions.

The differentiation of a proliferating CD4⁺ T cell into either aninflammatory T cell or a helper T cell is dependent on the cytokinesproduced by infectious agents, principally IL-12 and IL-4, the influenceof accessory molecules and on the nature of the MHC class II/peptidecomplex. As previously discussed, cell-mediated immunity involves thedestruction of intracellular pathogens by macrophages activated by Th1inflammatory cells directed primarily to intracellular parasitesincluding such parasites as Mycobacterium, Leishmania, Pneumocystis andthe like. In contrast, humoral immunity depends on the production ofantibody by B cells activated by helper T cells directed primarily atextracellular pathogens including Clostridium, Staphylococcus,Streptococcus, Polio virus, Pneumocystis and the like.

For example, recovery from certain types of infections, such asLeishmania, is associated with preferential production of IL-2/IFN-γ.Mice that mount a Th2 response to Leishmania fail to contain theinfection and ultimately die. Inappropriate production of cytokines ofthe Th2 type response has been frequently linked to allergic typediseases such as asthma and contact sensitivity.

Perhaps the strongest association of human disease with skewed patternsof cytokine production is the association of Th1 responses and Th1 typecytokines with autoimmune disease. Strong evidence in experimentalmodels indicates that many types of autoimmunity including diabetes,experimental models for multiple sclerosis, autoimmune thyroiditis, andthe like are mediated by Th1 type CD4⁺ T cells. The expression ofTh2-associated cytokines, such as IL-4, in these models interfere withthe development of autoimmune disease. Th2 type cytokines dampen theresponse of Th1 type cells while the Th1 type cytokines antagonize thedevelopment of Th2 type responses.

In view of the association of particular activated T cell subsets withparticular disease conditions, a need therefore exists to be able todirect the proliferation and activation of CD4⁺ T cells to a desired Tcell subset, a process that is extremely beneficial in altering thecourse of disease. One potential solution is to activate in vitro CD4⁺ Tcells that are first isolated from a subject who may optionally behaving either allergy or autoimmune conditions to produce cellssecreting a preferred cytokine profile. The resultant activated T cellsare then reintroduced to the subject to alter the course of disease andperhaps even provide a long term cure.

Alternative embodiments are directed at the ability to “vaccinate” apotentially responsive individual against the development of either aTh1 or Th2 response, whichever is applicable to that individual. Inother words, the selective induction of a particular T cell subset maybe achieved by inhibiting naive cells from developing toward theundersired phenotype. For example, in a potentially atopic individual,preventing a deleterious Th2 response would be beneficial as describedby Hetzel and Lamb, Clinical Immunol. Immunodath., 73:1-10 (1994).

In still a further embodiment, the compositions and methods of thisinvention are useful for actively stimulating the development of naive Tcells toward the desired phenotype. Existing therapeutic models to dateinclude the use of anti-cytokine antibodies are carrier proteins, theuse of idiotype/GM-CSF fusion protein vaccines to prolong effects ofexogenous cytokines, use of selected adjuvants, use ofliposome-encapsulated allergens, use of peptide analogs and the like asreviewed by Hetzel and Lamb, id. The authors, however, do state that forchronic Th2 responses to allergens in vivo, little experimental dataexists for the possibility of effecting a desired downregulation of theTh2 response.

In view of the foregoing, the compositions and methods of this inventionprovide a valuable means to accomplish the therapeutic interventionsdiscussed above.

The present invention allows one to define activation conditions thatreproducibly generate CD4⁺ T cell subsets that produce the desiredtherapeutic cytokine profile. Expression of particular cytokines islinked to a particular antigen presenting cell (APC) and theirassociated accessory molecules. Since both the cytokines produced by theAPC and the coordinately expressed accessory molecules are themselvesregulated by multiple factors, including the type of antigen, theaffinity of the T cell receptor (TCR)-antigen interaction, antigenconcentration and the like, predicting the outcome of T cell activationupon antigen presentation is historically very difficult. Indeed, asadditional accessory molecules have been proposed for the activationprocess in vivo, it has become increasingly clear that many diversemolecules are involved in the regulation of T cell responses and act incombinatorial fashion to effect the outcome of T cell activation.

The present invention provides the generation of synthetic APC thatpresent, in a neutral background, MHC class II molecules in combinationwith defined accessory molecules that are expressed preferably in anonmammalian insect cell. The advantage of using the insect cells as theexpression and presentation vehicles for the MHC class II/accessorymolecule compositions of this invention is that the cells do notendogenously produce regulatory cytokines and do not express mammalianaccessory molecules. This overcomes the inherent unpredictability ofusing mammalian APC that express many molecules that are capable ofaltering the T cell response. The present invention thus provides theability to isolate individual presenting molecules and accessorymolecules for expression in selected combinations that permitsreproducibility and predictability not available in other approaches.

2. Therapeutic Methods

As discussed above, the present invention relates to a method foractivating CD4⁺ T cells into differentiated armed effector T cellsubtypes. The method relates to providing a synthetic APC or matrixhaving anchored on the external surface a recombinant MHC class IIheterodimer that is capable of binding a peptide. The compositions alsohave at least one accessory molecule presented on the cell surface.Naive or activated CD4⁺ T cells can be obtained by removal from anindividual to be treated. The antigen presenting cells are thencontacted with the CD4⁺ T cells for a sufficient period of time toactivate the T cells into proliferating and differentiating into adesired T cell phenotype.

The activated CD4⁺ T-cells are separated from the cell line and put intoa suspension in an acceptable carrier and administered to theindividual.

It is preferred that human genes are used and, therefore, human moleculeanalogs are produced. As shown in prior U.S. Pat. No. 5,314,813, murinesystems provide particularly useful models for testing the operation ofT cell activation and demonstrate the applicability of the process forhuman systems. See also Sykulev et al., Immunity, 1:15-22 (1994).

a. Isolation of Resting or Activated CD4⁺ T Cells

Resting (or naive) as well as activated CD4⁺ cells that have not beenactivated to target a specific antigen presented in the context of MHCclass II are extracted from an individual for incubation or exposure tothe transformed cultures of the present invention. Naive cells can bedistinguished from primer cells primarily based on the cell surfacemarkers CD455RA and CD45.

It is also preferred that CD4⁺ cells are obtained from an individualprior to the initiation of other treatment or therapy which mayinterfere with the CD4⁺ cells' ability to be specifically activated. Forexample, if one is intending to treat an individual with an autoimmunedisease, it is preferable to obtain a sample of cells and culture priorto the initiation of adjunctive therapy such as steroid treatment orduring a window of time when the patient is not being treated at all.

When activating CD4⁺ T cells to alter the T cell-mediated immuneresponse in a patient, the patient is first analyzed for apatient-specific profile to assess the T cell phenotypic disease statefor instituting appropriate counter therapy involving production of theopposing T cell phenotype and cytokines. Cytokine profiles areestablished with anti-cytokine antibodies that are available from ATCCand by methods described in U.S. Pat. Nos. 5,405,751, 5,322,787 and5,209,920, the disclosures of which are hereby incorporated byreference. Preferred cytokine analyses include interleukin-2 (IL-2),interferon-γ (IFN-γ), tumor necrosis factor (TNF), interleukin-4 (IL-4),interleukin-10 (IL-10) and the like. As previously discussed, particularcytokine profiles are associated with T cell phenotypes and diseasestates.

In particular, where the condition is an autoimmune disease includingmultiple sclerosis, autoimmune thyroiditis, systemic lupuserythromatosus, myasthenia gravis, Crohn's disease and inflammatorybowel disease, the cytokine profile is produced by a Th1 type responsecharacterized by increased IL-2, IFN-γ and TNF. In contrast, where thecondition is an allergy, such as asthma and contact sensitivity, thecytokine profile is produced by a Th2 type response characterized byincreased IL-4 and IL-10.

After analyzing the patient cytokine profile and disease state,patient-isolated CD4⁺ T cells are contacted in vitro with the syntheticAPC, cell fragments or matrices of this invention as described below ina sufficient amount for a sufficient time to induce the contacted cellsto proliferate and differentiate into activated CD4⁺ T cells thatproduce a functionally opposing cytokine profile. That is, if thepatient were characterized as being a Th1 type responder, the antigen tobe presented to the patients CD4⁺ T cells would be that necessary toinduce the cells to proliferate and differentiate into a Th2 type. Theopposite treatment modality is performed if the patient is characterizedwith a Th2 type response. Thus, once the opposing activated cells arereturned to the patient as described below, the therapeutic goal ofeffecting an alteration in T cell phenotype response is attained.

Methods of extracting and culturing lymphocytes are well known. Forexample, U.S. Pat. No. 4,690,915 to Rosenberg describes a method ofobtaining large numbers of lymphocytes via lymphocytopheresis.Appropriate culturing conditions used are for mammalian cells, which aretypically carried out at 37° C.

Various methods are also available for separating put and/or enrichingcultures of CD4⁺ cells. Some examples of general methods for cellseparation include indirect binding of cells to specifically-coatedsurfaces. In another example, human peripheral blood lymphocytes (PBL),which include CD4⁺ cells, are isolated by Ficoll-Hypaque gradientcentrifugation (Pharmacia, Piscataway, N.J.). PBL lymphoblasts may beused immediately thereafter or may be stored in liquid nitrogen afterfreezing in FBS containing 10% DMSO (Sigma Chemical Co., St. Louis,Mo.), which conserves cell viability and lymphocyte functions.

Alternative methods of separating out and/or enriching cultures ofprecursor cells include both positive and negative selection procedures.For positive selection, after lymphocyte-enriched PBL populations areprepared from whole blood, subpopulations of CD4⁺ lymphocytes areisolated therefrom by affinity-based separation techniques directed atthe presence of the CD4 co-receptor antigen. These affinity-basedtechniques fluorescence-activated cell sorting (FACS), cell adhesion,magnetic bead separation and like methods. (See, e.g., Scher and Mage,in Fundamental Immunology, W. E. Paul, ed., pp. 767-780, River Press, NY(1984).) Affinity methods may utilize anti-CD4 co-receptor antibodies asthe source of affinity reagent. Alternatively, the natural ligand, orligand analogs, of CD4 receptor may be used as the affinity reagent.Various anti-T cell and anti-CD4 monoclonal antibodies for use in thesemethods are generally available from a variety of commercial sources,including the American Type Culture Collection (Rockville, Md.) andPharmingen (San Diego, Calif.).

Negative selection procedures are utilized to effect the removal ofnon-CD4 from the CD4⁺ population. This technique results in theenrichment of CD4⁺ cells from the T and B cell population ofleucophoresed patients. Depending upon the antigen designation,different antibodies may be appropriate. For example, monoclonalantibodies OKT4 (anti-CD4, ATCC No. CRL 8002) OKT 5 (ATCC Nos. CRL 8013and 8016), OKT 8 (anti-CD8, ATCC No. CRL 8014), and OKT 9 (ATCC No. CRL8021) are identified in the ATCC Catalogue of Cell Lines and Hybridomas(ATCC, Rockville, Md.) as being reactive with human T lymphocytes, humanT cell subsets, and activated T cells, respectively. Various otherantibodies are also available for identifying and isolating T cellspecies, including precursors and naive and activated memory matureperipheral T cells.

b. In Vitro Activation of CD4⁺ Cells

In order to optimize the in vitro conditions for the generation ofspecific CD4⁺ T cell phenotypes, the culture of antigen presenting cellsis maintained in an appropriate medium. Preferably, when using a supportof this invention that is an intact cell, the antigen-presenting cellsare Drosophila cells, which are preferably maintained in serum-freemedium (e.g. Excell 400). In alternative embodiments, however, when thesupport is a cell fragment or a matrix of an artificial support aspreviously described, the culture medium is selected to maintain theviability of the target cells.

Prior to incubation of the synthetic APC, cell fragments or matrices ofthis invention with the T cells to be activated, an amount of antigenicpeptide is provided to the APC or matrices in sufficient quantity tobecome loaded onto the human MHC class II molecules for expression onthe surface of the APC or matrices. As previously discussed, peptideloading can occur intracellularly or extracellularly. Both aspects areaccordingly encompassed in the activation process described herein butfor simplicity, loading of peptides is generically described as thedetails of peptide presentation to MHC class II heterodimers and loadinghave been previously discussed. Moreover, individual peptides as well aspeptide libraries are contemplated for use in preparing activated CD4⁺ Tcells also as previously described. According to the present invention,a sufficient amount of peptide is an amount that will allow about 200 toabout 500,000 and preferably about 200 to 1,000 or more, MHC class IImolecules loaded with peptide to be expressed on the surface of eachsynthetic APC or matrix. Preferably, the above compositions areincubated with 0.2 μg/ml up to 20 μg/ml peptide.

The isolated CD4⁺ cells are then incubated in culture with theappropriate peptide-loaded MHC class II heterodimers expressed onsynthetic APCs or matrices for a time period sufficient to activate CD4⁺cells. Preferably, the CD4⁺ cells shall thus be activated in anantigen-specific manner. The ratio of CD4⁺ cells to antigen-presentingcells may vary from individual to individual and may further depend uponvariables such as the amenability of an individual's lymphocytes toculturing conditions and the nature and severity of the diseasecondition or other condition for which the within-described treatmentmodality is used. Preferably, however, the lymphocyte:antigen-presentingcell or matrix ratio is preferably in the range of about 1:1 to 300:1.

The effector/antigen-presenting culture may be maintained for as long atime as is necessary to activate and enrich for a population of atherapeutically useable or effective number of CD4⁺ cells. In generalterms, the optimum time is between about one and five days, with amaximum specific level generally being observed after three to five daysof culture. In one embodiment of the present invention, in vitroactivation of CD4⁺ cells is detected within a brief period of time aftertransfection of a cell line.

Preferably, the activation of CD4⁺ cells is optimal within one week ofexposure to antigen-presenting cells. Thereafter, in a preferredembodiment, the activated CD4⁺ cells are further purified by isolationprocedures including density gradients, resetting with antibody-redblood cell preparations, column chromatography and the like. Followingthe purification, the resulting CD4⁺ cell preparation is furtherexpanded by maintenance in culture for a period of time to obtain apopulation of 10⁹ activated CD4⁺ cells. This period may vary dependingon the replication time of the cells but may generally be 14 days.

c. Separation of CD4⁺ Cells from Synthetic APC or Matrices

Activated CD4⁺ cells may be effectively separated from the antigenpresenting compositions of this invention using one of a variety ofknown methods. For example, monoclonal antibodies specific for the MHCclass II heterodimers, for the peptides loaded thereon, or for the CD4⁺cells (or a segment thereof) may be utilized to bind their appropriatecomplementary ligand. Antibody-tagged cells may then be extracted fromthe stimulator-effector cell admixture via appropriate means such as viawell-known flow cytometric or magnetic bead separation methods. Densitygradients can also be used to separate blast cells.

d. Therapeutic Treatment with Activated CD4 Cells

Effective amounts of the activated CD4⁺ cells can vary between in vitroand in vivo uses, as well as with the amount and type of target cellsexpressing the antigenic peptide used in activating the CD4⁺ population.The amount will also vary depending on the condition of the patient andshould be determined via consideration of all appropriate factors by thepractitioner. Preferably, however, about 1×10⁶ to about 1×10¹², morepreferably about 1×10⁸ to about 1×10¹¹, and even more preferably, about1×10⁹ to about 1×10¹⁰ activated CD4⁺ cells are utilized for adulthumans, compared to about 5×10⁶-5×10⁷ cells used in mice.

Preferably, as discussed above, the activated CD4⁺ cells are harvestedfrom the synthetic APC or matrix in culture prior to administration ofthe CDr⁺ cells to the individual being treated. It is important to note,however, that unlike other present and proposed treatment modalities, ina preferred embodiment, the present method uses a cell culture system ofDrosophila APC or acellular matrices that are not tumorigenic.Therefore, if complete separation of cells and activated CD4⁺ cells isnot achieved, there is no inherent danger known to be associated withthe administration of a small number of synthetic APC or matrices,whereas administration of mammalian tumor-promoting cells may beextremely hazardous.

Methods of re-introducing cellular components are known in the art andinclude procedures such as those exemplified in U.S. Pat. No. 4,844,893to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg. For example,administration of activated CD4⁺ cells via intravenous infusion isappropriate.

EXAMPLES

The following examples are intended to illustrate, but not limit, thepresent invention.

1. Preparation of pRmHa-3 Expression Vector

The pRmHa-3 expression vector for use in expressing MHC proteins inDrosophila Schneider 2 (S2) cells as described in this invention wasconstructed by ligating a Sph I-linearized pRmHa-1 DNA expression vectorwith a DNA fragment resulting from a Sph I restriction digest of apRmHa-2 expression vector to form the pRmHa-3 expression vector asdescribed below. The ligating of the linearized pRmHa-1 with the pRmHa-2fragment in this manner was performed to remove one of two Eco RIrestriction endonuclease cloning sites present in pRmHa-1. Thus, theresultant pRmHa-3 expression vector contained only one Eco RIrestriction site in the multiple cloning site (polylinker) into whichvarious MHC class II-encoding DNA fragments were inserted as describedin the Examples.

A. Preparation of pRmHa-1 Expression Vector

The pRmHa-1 expression vector, containing a metallothionein promoter,metal response consensus sequences (designated MT) and an alcoholdehydrogenase (ADH) gene containing a polyadenylation signal isolatedfrom Drosophila melanogaster, was constructed as described by Bunch etal., Nucl. Acids Res., 16:1043-61 (1988). The plasmid expression vector,pUC18, having the ATCC accession number 37253, was used as the sourcevector from which subsequent vectors described herein were derived. ThepUC18 plasmid contains the following restriction sites from 5′ to 3′ inthe multiple cloning site, all of which are not illustrated in theschematic representations of the pUC18-derived vectors in FIG. 1: EcoRI; Sac I; Kpn I; Sma I and Sma I located at the same position; Bam HI;Xba I; Sal I, Acc I and Hinc II located at the same position; Pst I; SphI and Hind III. The pUC18 vector was first digested with Hind III toform a linearized pUC18. Blunt ends were then created by filling in theHind III ends with DNA polymerase I large fragment as described byManiatis et al., Molecular Cloning: A Laboratory Manual, eds. ColdSpring Harbor Laboratory, New York (1982).

The resultant linearized blunt-ended pUC18 vector was ligated with a 740base pair (bp) Hinf I fragment from the Drosophila melanogaster ADH genecontaining a polyadenylation signal. The ligated ADH allele was firstisolated from the plasmid pSACI, described by Goldberg et al., Proc.Natl. Acad. Sci. USA, 77:5794-5798 (1980), by digestion with Hinf Ifollowed by blunt ending with Klenow resulting in the nucleotidesequence listed in SEQ ID NO 1. The PSACI vector containing the ADHallele was constructed by subcloning into pBR322 (ATCC accession number31344) a 4.7 kilobase (kb) Eco RI fragment of Drosophila DNA selectedfrom a bacteriophage lambda library containing random, high molecularweight (greater than 15 kb). The 5′ Hinf I restriction site occurrednaturally in the ADH gene at position 1770 as described by Kreitman,Nature, 304:412-417 (1983). The 3′ Hinf I site was derived from thepUC18 vector into which the ADH gene had been cloned. This position wasfour bases 3′ to the Xba I site at position 2500 of the ADH gene. TheADH segment extended from 35 bp upstream of the polyadenylation/cleavagesequence in the 3′ untranslated portion of the ADH mRNA to 700 bpdownstream of the polyadenylation signal. The resultant pUC18-derivedvector containing the ADH gene fragment was designated pHa-1 as shown inFIG. 1A.

A 421 bp Eco RI/Stu I MT gene fragment for insertion into pHa-1 wasobtained from a clone containing DNA of approximately 15.3 kb in aDrosophila melanogaster genomic DNA library. The library, prepared witha Mbo I partial digestion of imaginal DNA, was cloned in the lambdaderivative EMBL4. The 421 bp fragment contained the MT promoter andmetal response consensus elements of the Drosophila MT gene (Maroni etal., Genetics, 112:493-504 (1986)). This region, containing the promoterand transcription start site at nucleotide position +1, corresponded toposition −370 to nucleotide position +54 of the MT gene (SEQ ID NO 2).The resultant fragment was then ligated into pHa-1 expression vectorprepared above that was previously linearized with Eco RI and Sma I. The3′ blunt end in MT created by the Stu I digest was compatible with theblunt end in pHa-1 created by the Sma I digest. The resultantpUC18-derived vector containing a 5′ Drosophila MT gene fragment and a3′ ADH gene fragment was designated pRmHa-1. The pRmHa-1 expressionvector contained the origin of replication (ori) and the beta-lactamasegene conferring resistance to ampicillin (Amp^(r)) from pUC18 as shownin FIG. 1A on the pHa-1 vector. The pRmHa-1 also contained from 5′ to 3′the MT gene fragment, the multiple cloning site and the ADH genefragment. The pRmHa-1 vector was used as described below in theconstruction of the pRmHa-3 expression vector.

B. Preparation of pRmHa-2 Expression Vector

For constructing the pRmHa-2 expression vector shown in FIG. 1A, the MTfragment prepared above was inserted into the pUC18-derived vector pHa-1as described for constructing pRmHa-1 above with a few modifications. AnEco RI linker was added to the Stu I site of the Eco RI/Stu I-isolatedMT gene fragment prepared above to form a metallothionein fragmenthaving Eco RI restriction sites on both ends. The resultant fragment wasthen ligated into the ADH fragment-containing pUC18 expression vectorthat was previously linearized with Eco RI. The resultant pUC18-derivedvector containing a 51 Drosophila MT gene fragment and a 3′ ADH genefragment having two Eco RI restriction sites 5′ to the multiple cloningsite was designated pRmHa-2. The pRmHa-2 expression vector contained theorigin of replication (ori) and the beta-lactamase gene conferringresistance to ampicillin (AMp^(r)) from pUC18. The diagram of pRmHa-2also shows the 5′ to 3′ contiguous positions of the MT gene fragment,the multiple cloning site and the ADH gene fragment. The pRmHa-2 vectorwas used along with pRmHa-1 as described below in the construction ofthe pRmHa-3 expression vector.

C. Preparation of pRmHa-3 Expression Vector

To prepare the pRmHa-3 expression vector that had only one Eco RIrestriction site, a fragment from pRmHa-2 was ligated into pRmHa-1. Forthis construction, pRmHa-2, prepared above, was first digested with SphI. The resultant Sph I fragment beginning in the middle of the MT geneand extending to the Sph I site in the multiple cloning site was firstisolated from the pRmHa-2 vector and then ligated into pRmHa-1 that waspreviously modified to remove the Eco RI restriction site 5′ to the MTgene fragment then linearized with Sph I. This process is schematicallyillustrated in FIG. 1B. To remove the Eco RI site in pRmHa-1, the vectorwas first digested with Eco RI to form a linearized vector, then bluntended with Mung Bean nuclease and religated.

A schematic of the pRmHa-3 vector is shown in FIG. 1C. The relativepositions of the various restriction sites from the pUC18 vector fromwhich pRmHa-3 was derived are indicated in the figure. The pRmHa-3vector, being derived from pUC18, contains the pUC18 origin ofreplication and beta-lactamase gene conferring ampicillin resistance.Thus, MHC class II-encoding DNA fragments as prepared in this inventionand cloned into the multiple cloning site of pRmHa-3 weretranscriptionally regulated by the MT promoter and polyadenylated viathe ADH gene.

2. Preparation and Expression of Expressible MHC Class II Genes

A. Amplification and Expression of MHC Class II Genes

Genes or cDNAs encoding any preferred mammalian MHC Class II are clonedvia use of the polymerase chain reaction (PCR). The primers describedherein are used to amplify the appropriate Class II cDNAs in separatereactions which are then cloned and sequenced. To create MHC class IIproteins, full length murine IA^(d) α- and β-chain cDNAs were firstobtained followed by PCR amplification and modification. The completenucleotide sequences of the murine IA^(d) α-chain cDNA is described byBenoist et al., Cell, 34:169-177 (1983) and is also listed in GenebankAccession Number K01923. The complete nucleotide sequences of the murineIA^(d) β-chain cDNA is described by Malissen et al., Science,221:750-754 (1983) and is also listed in GenBank Accession NumbersK00007 and K00008. To amplify each chain, mouse splenocytes were used asa source of total RNA. First strand cDNA was synthesized by usingoligo(dT) and avian myeloblastosis virus reverse transcriptase. Theresulting cDNA was used in a PCR amplification reaction utilizing theappropriate primers described below and a GeneAmp kit and thermal cycler(Perkin-Elmer/Cetus, Norwalk, Conn.). Reaction conditions preferablycontained 1 μg cDNA template and 200 nM of each oligonucleotide primer.Thirty cycles were run as follows: (a) 1 minute at 92° C.; (b) 1 minuteat 60° C.; and (c) 1 minute at 72° C. The PCR reaction was then heatedto 99° C. for 10 minutes to inactivate the Taq polymerase and the endsof the DNA were made blunt by T4 polymerase (Stratagene, La Jolla,Calif.).

For all the oligonucleotide primers indicated in the Examples, the 5′primer is also referred to as the forward primer or sense primer as ithas the same sequence as the top strand of the cDNA for hybridizing tothe complementary bottom strand. In contrast, the 3′ primer is alsoreferred to as the backward primer or anti-sense primer as it has thesame sequence as the bottom strand of the cDNA for hybridizing to thecomplementary top strand. Both primers are written in the 5′ to 3′direction. The full length IA^(d) α-chain cDNA was amplified with the 5′primer having the nucleotide sequence5′CTTGAATTCCACCATGCCGTGCAGCAGAGCTCTGA3′ (SEQ ID NO 3). The 5′ primer wasalso designed with an Eco RI restriction site to allow for directionalligation of the amplified products into recipient expression vectors.The 3′ primer 5′TTTGGATCCTCATAAAGGCCCTGGGTGTC3′ (SEQ ID NO 4) was alsodesigned to contain a Bam HI restriction site.

The full length IA^(d) β cDNA was amplified with the 5′ forward primerhaving the nucleotide sequence 5′CTTGAATTCCACCATGGCTCTGCAGATCCCCA3′ (SEQID NO 5). The 5′ primer was also designed with an Eco RI restrictionsite to allow for directional ligation of the amplified products intorecipient expression vectors. The 3′ primer5′TTTGGATCCTCACTGCAGGAGCCCTGCT3′ (SEQ ID NO 6) was designed to contain aBam HI restriction site. The modified cDNAs encoding the murine IA^(d)α- and β-chains were first separately directionally cloned into thepolylinker at the Eco RI and Bam HI restriction sites of themetallothionein promoter-driven pRmHa-3 vector, then sequenced by dyeterminator technique on an Applied Biosystem 373A automated sequencer.

The complete nucleotide sequences of the murine IA^(d) α- and β-chainamplified regions cloned into pRmHa-3 are respectively listed in SEQ IDNOs 7 and 8.

The separate plasmids were then transfected into Drosophila melanogasterSchneider-2 cells (ATCC CRL 10974, Rockville, Md.) as describedelsewhere (Jackson et al., Proc. Natl. Acad. Sci. U.S.A., 89;12117-211992). Equal amounts of the plasmids encoding the two class II chainswere cotransfected together with a neomycin resistance gene, plasmidphshsneo (Bunch et al., Nuc. Acids Res., 16:1043-1061 (1988) at a ratioof 1:30 to produce stable cell lines that were derived by G418 selectionin Schneider's Drosophila medium (Gibco/BRL, Grand Island, N.Y.)supplemented with 10% fetal calf serum (heat treated for 1 hour at 55°C.), 100 units/ml penicillin, 100 μg/ml streptomycin, and 1 mM glutamineover a period of 4 weeks. Specifically, after transfection, thesupernatant was carefully removed and the cells were transferred to a 75cm² flask in a total volume of 12 ml Schneider medium containing 500μg/ml Geneticin (G418) (Gibco/BRL, Grand Island, N.Y.). After 4 days, 4ml of the culture were transferred to a fresh flask containing 6 ml ofSchneider medium with 500 μg/ml G418. This procedure was repeated every4-7 days until a stable population of cells emerged which weakly adheredto the flask and grew with a doubling time of approximately 24 hours.These cells were subsequently cultured and passaged in the selectionmedia as described above. Frozen aliquots of the stably transfectedcells were prepared by collecting 5-20×10⁶ cells by centrifugation andresuspending them in 1 ml of cell freezing media (93% fetal calfserum/7% dimethylsulfoxide). Aliquots were then placed at −70° C. for 1week and subsequently transferred to liquid nitrogen storage. Expressionof the stably transfected MHC class II IA^(d) α- and β-chain genes wasinduced with 0.7 mM cupric sulfate for 24 hours at 27° C.

Expression of MHC class II IA^(d) heterodimers at the cell surface ofthe transfected Drosophila cells was evaluated by flow cytometry afterstaining with with MKD6, an IA^(d)-specific monoclonal antibody (Kappleret al., J. Exp. Med., 153:1198 (1981). Briefly, aliquots of cells(5×10⁵) were transferred into tubes on ice, collected by centrifugation(1,000×g for 5 minutes), resuspended in 0.1 ml of Drosophila medium with5% horse serum containing the appropriate primary antibody (MKD6). Aftera 20 minute incubation at room temperature, cells were washed twice in 3ml of Schneider's medium containing horse serum and resuspended in 0.1ml of medium containing FITC-labeled secondary antibody (Cappell,Durham, N.C.). After a 20 minute incubation on ice, cells were washedtwice with Schneider's medium containing horse serum and resuspended inthis buffer at a concentration of 1×10⁶/ml. Propidium iodide was addedto permit exclusion of dead cells from the analysis. Samples were thenanalyzed by FACScan or FACSort instrument (Becton Dickinson). Syntheticantigen presenting cells of this invention that express the murineIA^(d) MHC class II haplotype, described herein and well known in theart, with one or more accessory molecules were produced as describedbelow.

Full-length human MHC class II α- and β-chains for the DR, DQ and DPhaplotypes are amplified from peripheral blood cells which include Bcells, macrophages and dendritic cells. The 5′ and 3′ primers used foramplifying each of the haplotypes have the following sequences:

DR α: 5′ primer = 5′CCACCATGGCCATTAGTGGAGTC3′ (SEQ ID NO 9) 3′ primer =5′TTTGGATCCTTACAGAGGCCCCCTGCGTT3′; (SEQ ID NO 10) DR β: 5′ primer =5′CCACCATGGTGTGTCTGAGGCTCC3′ (SEQ ID NO 11) 3′ primer =5′TTTGGATCCTCAGCTCAGGAATCCTCTTG3′; (SEQ ID NO 12) DQ α: 5′ primer =5′CCACCATGGTCCTAAACAAAGCTCTGAT3′ (SEQ ID NO 13) 3′ primer =5′TTTGGATCCTCACAAGGGCCCTTGGTGTCT3′; (SEQ ID NO 14) DQ β: 5′ primer =5′CCACCATGGCTTGGAAGAAGGCCTTT3′ (SEQ ID NO 15) 3′ primer =5′TTTAGATCTCAGTGCAGAAGCCCTTT3′; (SEQ ID NO 16) DP α: 5′ primer =5′CCACCATGGGCCCTGAAGACAGAAT3′ (SEQ ID NO 17) 3′ primer =5′TTTGGATCCTCACAGGGTCCCCTGGGC3′; (SEQ ID NO 18) DP β: 5′ primer =5′CCACCATGGTTCTGCAGGTTTCTGCG3′ (SEQ ID NO 19) 3′ primer =5′TTTGGATCCTTATGCAGATCCTCGTTGAA3′. (SEQ ID NO 20)

The amplification conditions for obtaining the above human haplotypesare identical to those described above for the murine counterparts.

Synthetic antigen presenting cells of this invention that express ahuman MHC class II haplotypes, described herein and well known in theart, with one or more accessory molecules are produced as describedbelow.

B. Amplification of Invariant and HLA-DM Chain cDNAs

For some aspects of the present invention as described in Section B inthe detailed description, the invariant chain and HLA-DM α and HLA-DM βare co-expressed with a selected MHC class II heterodimer and at leastone accessory molecule described above. In this instance, the MHC classII α- and β-chains, invariant chain and HLA-DM α- and β-chains aretransfected into recipient antigen presenting cells in molar ratios5:5:8:1:1. The ratio of accessory molecules to the molar ratio of theother genes is 1:1. The resultant transfected cells are then induced forexpression of the exogenous genes as described above to generate asynthetic antigen presenting cell line of this invention.

1) Invariant Chain

The murine invariant chain cDNA is generated from mouse spleen cells.The full-length invariant chain is amplified from this source with theprimer pair of a 5′ and 3′ primer having the respective nucleotidesequences 5′AAGAATTCACTAGAGGCTAGAGCCAT3′ (SEQ ID NO 21) and5′AAGGATCCTCACAGGGTGACTTGACC3′ (SEQ ID NO 22). As described above, the5′ and 3′ primers were designed to respectively incorporate Eco RI andBam HI restriction sites. Following cloning of the amplified invariantchain into a Eco RI/Bam HI-digested pRmHa-3 vector, the resultant clonewas sequenced and determined to have the sequence listed in SEQ ID NO23.

The human invariant chain cDNA used in this invention is generated usingRNA from γ-interferon-induced HeLa cells (ATCC Accession Number CCL 2).The resultant cDNA is then used as a template in PCR with the 5′ and 3′oligonucleotide primers having the respective nucleotide sequences5′AAGAATTCACCATGGATGATCAGCGCGACCTT3′ (SEQ ID NO 24) and5′AAAGGATCCTCACATGGGGACTGGGCCCAGA3′ (SEQ ID NO 25). The resulting PCRfragments are cleaved with Eco RI and Bam HI before ligation intosimilarly digested pRmHa-3.

2) HLA-DM

Messenger RNA from γ-interferon-induced HeLa cells is also used tosynthesize the α- and β-chains of HLA DM cDNA that is then used as atemplate in PCR. The α-chain is amplified with the 5′ and 3′ primer pairhaving the respective nucleotide sequences5′AAACCATGGGTCATGAACAGAACCA3′, (SEQ ID NO 26) and5′TTTGTCGACTCAGTCACCTGAGCAAGG3′ (SEQ ID NO 27). The β-chain is amplifiedwith the 5′ and 3′ primer pair having the respective nucleotidesequences 5′AAACCATGGTCTCATTCCTGCC3′ (SEQ ID NO 28) and5′TTTGTCGACCTAGGAAATGTGCCATCC3′ (SEQ ID NO 29). The resulting PCRfragments are cleaved with Nco I and Sal I before separate ligation intoa similarly digested pRmHa-3.

C. Amplification of Genes Encoding Accessory Molecules

1) Adhesion Molecules

a. ICAM-1, ICAM-2, and ICAM-3

For isolating murine ICAM-1, spleen cells were isolated from Balb/cmice. The spleen cells were first stimulated with conA before isolationof mRNA using the FastTrack kit (Invitrogen, San Diego, Calif.)according to the manufacturers' instructions. cDNA was synthesized fromthe mRNA as described above. The resultant cDNA was then subjected toPCR with the following respective 51 and 3′ primers that were designedbased on the published cDNA nucleotide sequence (Siu, G. et al., J.Immunol., 143:3813-3820 (1989)): 5′TTTAGAATTCACCATGGCTTCAACCCGTGCCAAG3′(SEQ ID NO 30) and 5′TTTAGTCGACTCAGGGAGGTGGGGCTTGTCC3′ (SEQ ID NO 31).The PCR products were then cleaved with the restriction enzymes Eco RIand Sal I and ligated into a similarly digested pRmHa-3.

The above expression construct was then co-transfected into DrosophilaS2 cells with the murine IA^(d) α- and β-chain genes prepared aboveusing the calcium phosphate method as described above in the molar ratioof 1:1:1 of ICAM-1:α-chain:β-chain. Stably transfected cells wereobtained as described above. Synthetic antigen presenting Drosophila S2cells expressing the accessory molecule ICAM-1 with the IA^(d) α- andβ-chains on the surface of the cell were then produced by inducingexpression as previously described.

In other embodiments, synthetic antigen presenting cells containinggenes encoding ICAM-1, B7.1 and/or B7.2 in conjunction with the murineIA^(d) molecules were also produced for generating synthetic antigenpresenting cells as used in activation assays as described in Example 5.

Human ICAM-1 is similarly amplified from mRNA isolated from the humancell line K562, originated from human chronic myelogenous leukemia (ATCCAccession Number CCL-243) and cultured under recommended conditions(i.e., RPMI with 10% fetal calf serum at 37° C. with 5% CO₂). The PCRprimers for amplifying the human accessory molecules are designed basedon known available sequences and in consideration of the 5′ and 3′cloning sites needed to clone into the appropriate vectors. Thenucleotide sequence of human ICAM-1 cDNA is available through GenBankAccession Number GB J03132. The ICAM-1 5′ and 3′ primers have therespective nucleotide sequences

5′ACCCTTGAATTCATGGCTCCCAGCAGCCCCCGGCCC3′ (SEQ ID NO 32) and5′ATTACCGGATCCTCAGGGAGGCGTGGCTTGTGTGTTCGG3′. (SEQ ID NO 33)

PCR is performed with these primers as previously described to obtainamplified human ICAM-1. The resultant PCR products are then cloned intopRmHa-3 as previously described followed by transfection into recipientcells, also as previously described.

Similarly, human ICAM-2 and ICAM-3, the nucleotide sequences of whichare available with the respective GenBank Accession Numbers GB X15606and GB S50015, are amplified with comparable primer pairs. Therespective 5′ and 3′ primers for amplifying ICAM-2 have the nucleotidesequence 5′AAGGTACCCGTGGAGACTGCCAGAGAT3′ (SEQ ID NO 34) and5′TTTGGATCCCTATGGCCGGAAGGCCTG3′ (SEQ ID NO 35). The respective 5′ and 3′primers for amplifying ICAM-3 have the nucleotide sequence5′AAGAATTCCTGTCAGAATGGCCACCAT3′ (SEQ ID NO 36) and5′TTTAGATCTTCACTCAGCTCTGGACGGT′ (SEQ ID NO 37). The resultant amplifiedICAM products are then separately ligated into pRmHa-3 and separatelytransfected into Drosophila S2 cells.

Synthetic antigen presenting Drosophila S2 cells expressing theaccessory molecule ICAM-1 with a selected human MHC class II haplotypeon the surface of the cell are then produced by inducing expression aspreviously described.

In other embodiments, synthetic antigen presenting cells containinggenes encoding ICAM-1, ICAM-2 or ICAM-3 in combination with B7.1 and/orB7.2 along with a human haplotype are also produced for generatingsynthetic antigen presenting cells as used in activation assays asdescribed in Example 5. Additional permutations contemplated for use inthis invention include the above combinations with LFA-3 and/or Fasligand (FasL), both of which are other accessory molecules as describedbelow.

b. LFA-3

Human LFA-3 is isolated from blood lymphocytes. The nucleotide sequenceof human LFA-3 cDNA is available through GenBank Accession Number GB109083. Human LFA-3 is amplified accordingly with a 5′ and 3′ primerhaving the respective nucletide sequences

5′ACCCTTGAGCTCATGGTTGCTGGGAGCGACGCGGGG3′ (SEQ ID NO 38) and5′ATTACCGGATCCTTAAAGAACATTCATATACAGCACAATACA3′. (SEQ ID NO 39)

As described previously, synthetic antigen presenting cells containinggenes encoding human LFA-3 alone or in various combinations with theother accessory molecules described herein along with a human haplotypeare also produced for generating synthetic antigen presenting cells asused in activation assays as described in Example 5.

2) Costimulatory Molecules

a. B7.1

cDNA was generated from mRNA isolated from mice as described above forICAM-1. The resultant cDNA was then subjected to PCR with the followingrespective 5′ and 3′ oligonucleotide primers shown in the 5′ to 3′direction that were designed based on the published cDNA nucleotidesequence (Freeman, et al., J. Exp. Med., 174:625-631 (1991)):5′TTTAGAATTCACCATGGCTTGCAATTGTCAGTTG3′ (SEQ ID NO 40) and5′TTTAGTCGACCTAAAGGAAGACGGTCTGTTC3′ (SEQ ID NO 41). The PCR productswere cleaved with the restriction enzymes Eco RI and Sal I and ligatedinto a similarly digested pRmHa-3.

The above expression construct was then co-transfected into DrosophilaS2 cells with the murine IA^(d) α- and β-chain genes prepared aboveusing the calcium phosphate method as described above in the molar ratioof 1:1:1 of B7.1:α-chain:β-chain. Stably transfected cells were obtainedas described above. Synthetic antigen presenting Drosophila S2 cellsexpressing the accessory molecule B7.1 with the IA^(d) α- and β-chainson the surface of the cell were then produced by inducing expression aspreviously described.

In other embodiments, synthetic antigen presenting cells containinggenes encoding murine ICAM-1, B7.1 and/or B7.2 in conjunction with themurine IA^(d) molecules are also produced for generated syntheticantigen presenting cells as used in activation assays as described inExample 5.

Human B7.1 is similarly isolated from K562 cells and cloned into pRmHa-3as described above. The nucleotide sequence of human B7.1 cDNA isavailable through GenBank Accession Number GB M83071. The 5′ and 3′primers have the respective nucleotide sequences,

5′ACCCTTGAATCCATGGGCCACACACGGAGGCAG3′ (SEQ ID NO 42) and5′ATTACCGGATCCTTATACAGGGCGTACACTTTCCCTTCT3′. (SEQ ID NO 43)The resultant PCR products are then inserted into pRmHa-3 that is thenco-transfected along with amplified cloned human haplotype genes intoDrosophila S2 cells.

Synthetic antigen presenting Drosophila S2 cells expressing theaccessory molecule B7.1 with a selected human MHC class II haplotype onthe surface of the cell are then produced by inducing expression aspreviously described.

In other embodiments, synthetic antigen presenting cells containinggenes encoding human B7.1 in various combinations with the otheraccessory molecules described herein along with a human haplotype arealso produced for generating synthetic antigen presenting cells as usedin activation assays as described in Example 5.

b. B7.2

Murine IC-21 cells (ATCC Accession Number TIB 186) were propagated inRPMI 1640 medium containing 10% fetal calf serum. cDNA was synthesizedfrom the mRNA isolated from these cells as described above. Theresultant cDNA was then subjected to PCR with the following respective5′ and 3′ oligonucleotide primers shown in the 5′ to 3′ direction thatwere designed based on the published cDNA nucleotide sequence (Freeman,et al., J. Exp. Med., 178:2185-2192 (1993)):5′TTTAGAATTCACCATGGACCCCAGATGCACCATGGG3′ (SEQ ID NO 44) and5′TTTAGTCGACTCACTCTGCATTTGGTTTTGCTGA3′ (SEQ ID NO 45). The PCR productswere cleaved with the restriction enzymes Eco RI and Sal I and ligatedinto a similarly digested pRmHa-3.

The above expression construct was then co-transfected into DrosophilaS2 cells with the murine IA^(d) α- and β-chain genes prepared aboveusing the calcium phosphate method as described above in the molar ratioof 1:1:1 of B7.2:α-chain:β-chain. Stably transfected cells were obtainedas described above. Synthetic antigen presenting Drosophila S2 cellsexpressing the accessory molecule B7.2 with the IA^(d) α- and β-chainson the surface of the cell were then produced by inducing expression aspreviously described.

In other embodiments, synthetic antigen presenting cells containinggenes encoding murine B7.2 in conjunction with ICAM-1 and the murineIA^(d) molecules were also produced following the procedures describedabove for generated synthetic antigen presenting cells as used inactivation assays as described in Example 5.

Human B7.2 is isolated from the human cell line HL60, that originatedfrom a human promyelocytic leukemia (ATCC Accession Number CCL-240). Thenucleotide sequence of human B7.2 cDNA is available through GenBankAccession Number GB M83071. The 5′ and 3′ primers for amplifying humanB7.2 have the respective nucleotide sequences

5′ACCCTTGAGCTCATGGATCCCCAGTGCACTATG3′ (SEQ ID NO 46) and5′ATTACCCCCGGGTTAAAAACATGTATCACTTTTGTCGCATGA3′. (SEQ ID NO 47)

The amplified B7.2 products are then cloned into pRmHa-3 as previouslydescribed for tranfection into Drosophila S2 cells along with constructsfor a selected human haplotype. Expression of the transfected genes isinduced as previously described.

In other embodiments, synthetic antigen presenting cells containinggenes encoding human B7.2 in various combinations with the otheraccessory molecules described herein along with a human haplotype arealso produced for generating synthetic antigen presenting cells as usedin activation assays as described in Example 5.

3. Survival Molecules

Human Fas ligand is isolated from activated human T cells. Thenucleotide sequence of human Fas ligand cDNA is available throughGenBank Accession Number GB U08137. Human Fas ligand is amplifiedaccordingly with a 5′ and 3′ primer pair having the respectivenucleotide sequences

5′AAAGGATCCACCATGCAGCAGCCCTTCAATT3′ (SEQ ID NO 48) and5′TTTGGATCCTTAGAGCTTATATAAGCCGA3′. (SEQ ID NO 49)

Human CD70, the ligand for CD27 expressed on T cells, is amplified asdescribed above with a 5′ and 3′ primer pair having the respectivenucleotide sequences

5′AAGAATTCGGTACCATGCCGGAGGAGGGTTCGG3′ (SEQ ID NO and 50)5′TTTGGATCCTCAGGGGCGCACCCACTGCA3′. (SEQ ID NO 51)

As described previously, synthetic antigen presenting cells containinggenes encoding human Fas ligand alone or in various combinations withthe other accessory molecules described herein along with a humanhaplotype are also produced for generating synthetic antigen presentingcells as used in activation assays as described in Example 5.

3. Preparation of MHC Class II Anchored on Synthetic Supports

The examples described herein present a new method to immobilize highamounts of MHC class II molecules and single accessory molecules orvarious combinations thereof on various surfaces (fly cells, red bloodcells, latex beads) in native conformation as judged by monoclonalantibody binding and resetting experiments (T cell receptor binding).This method can be extended to other synthetic surfaces includingartificial phospholipid membranes. Phosphatidyiethanolamine as well asavidin-coupled phospholipids are particularly relevant this invention.These phospholipids are commercially available from Lipex BiomembraneInc., Vancouver, BC, Canada.

A. Immobilization of Biotinylated MHC Class II on Avidin-Coated RedBlood Cells

NHS-LC-biotin, neutravidin and biotin-BMCC are purchased from Pierce(Rockford, Ill.). Sheep red blood cells are obtained from the ColoradoSerum Company (Denver, Colo.). Drosophila S2 cells expressing MHC classII are prepared as described above. Monoclonal antibodies MKD6 andM5-114 (ATCC Accession Number TIB 120 for the hybridoma secreting MKD6)are used as hybridoma cell culture supernatants. The protocol used isdescribed by Muzykantov and Taylor (Anal. Biochem., 223:142-148 (1994)).Briefly, SRBC are washed four times in PBS, biotinylated usingNHS-LC-biotin, washed again 4 times in PBS, incubated with neutavidin,and finally washed four times and stored at 4° C. in PBS containing 3%fetal calf serum and 0.02% sodium azide. Recombinant expressed MHC classII α- and β-chains are biotinylated using biotin-BMCC, amaleimide-coupled biotin which reacts with thiol groups. Biotinylationis performed as recommended by the manufacturer. Unreacted biotin isremoved using Centricon 10.

Biotinylated MHC class II α- and β-chains are immobilized by incubationat a final concentration of 0.2 mg/ml with avidin-coated SRBC for 30minutes followed by washing in DMEM containing 10 fetal calf serum. SRBCwith attached MHC class II are used immediately.

Immobilization of biotinylated MHC class II α- and β-chains onavidin-coated SRBC is done as indicated above. Attachment is assessedusing flow cytofluorometry using the antibodies described above. Forresetting assays, either Drosophila S2 cells expressing MHC class II orMHC class II-coated SRBC are incubated with specific peptide (seeExample 4) (0.02 mM) or an irrelevant peptide (0.02 mM) for 30 minuteson ice; CD4⁺ T cells are then added, the proportion being 10 T cells forone Drosophila S2 cell, or 10 SRBC for 1 T cell. The mixture is thenpelleted and kept on ice for at least 30 minutes. Cells are thencarefully resuspended and rosettes are counted, a rosette being aDrosophila S2 cell bound to at least 3 CD4⁺ T cells, or a CD4⁺ T cellbound to at least 3 SRBC.

B. Immobilization of Biotinylated MHC Class II on Avidin-Coated LatexBeads

Six micron diameter latex sulfate beads are purchased from InterfacialDynamics Corporation (Portland, Oreg.) and biotinylated according to theprotocol described above. Avidin-coated latex beads are prepared using a1% suspension of the latex beads incubated in PBS containing 1 mg/ml ofneutravidin for 1 hour at room temperature. An equal volume of PBScontaining 10% fetal calf serum is then added. After 1 hour ofincubation at room temperature, the beads are washed 3 times and usedfor binding of recombinant biotinylated MHC class II.

Recombinant biotinylated MHC class II is immobilized by incubation at afinal concentration of 0.2 mg/ml with avidin-coated latex beads for 30minutes followed by washing in DMEM containing 10% fetal calf serum.SRBC with attached MHC class II are used immediately. Rosettingexperiments are performed as previously described.

C. Immobilization and Detection of MHC Class II Bound to Various SolidSupports Such as Plastic Microwell Plates

The MHC class II molecules are immobilized by direct binding tomicrotiter plates (Corning) and detected as follows: MHC class II isdiluted to a desired concentration in PBS, e.g. 1 mg/ml for 100 ng/well,and then added to each well on the plastic microtiter plate. The plateis incubated for 1 hour at room temperature. After incubation, the plateis washed once with PBS and 200 μl 2% bovine serum albumin (BSA) inPBS+(0.05%) and Tween (PBST) is added followed by incubation for anotherhour at room temperature. The plate is washed 3 times with PBST andbiotinylated anti-MHC class II monoclonal antibody was added (1:2500) in2% BSA in PBS. The plate is incubated another hour at room temperatureand washed 3 times with PBST. Avidin conjugated HRP is added (1:2500) in2% BSA in PBS. Following another hour of incubation at room temperature,the plate is washed 3 times with PBST and H₂O₂ or thophenyldiamine wasadded. The reaction is stopped with H₂SO₄. Reaction product is detectedcalorimetrically at 490 nm. Recombinant MHC class II molecules canalternatively be bound through biotin-avidin linked interactions withthe substrate. In this embodiment, the microwell plates are coated with100 μl avidin diluted in PBS to a concentration of 0.001 mg/ml. Excessavidin is removed by a PBS wash. The above procedure for presenting anddetecting MHC class II binding is followed.

Recombinant MHC molecules are alternatively immobilized by a linkagebased on a poly-histidine tag added to the MHC interacting with thenickel bound to the substrate.

The above procedure for binding and detection is followed using nickelchelate coated microwell plates (Xenopore) and expressed recombinant MHCmolecules with a poly-histidine tag.

4. Peptide Generation

Antigenic peptides according to the present invention are obtained fromnaturally-occurring sources, including those described in thespecification, or may be synthesized using known methods. In variousexamples disclosed herein, all peptides were separately chemicallysynthesized by T-BOC chemistry on an Applied Biosystem instrument, ABI431A (Foster City, Calif.), and purified on a C18 reverse phase column.Isolation or synthesis of peptides generated from a random assortment ofamino acid residues may also be appropriate, particularly when one isattempting to ascertain a particular epitope in order to load an emptyMHC molecule with a peptide most likely to stimulate CD4 cells. Mixturesof randomized peptides are obtained by using proteasomes, by subjectinga protein or polypeptide to a degradative process, such as digestionwith chymotrypsin, or by synthesis.

While the cell lines of the present invention are able to degradeproteins and polypeptides into smaller peptides capable of being loadedonto human MHC class II molecules, it is preferable to introduce thepeptides directly into the cell culture to facilitate a more rapidloading and expression process.

If peptides are synthesized having randomly incorporated amino acidresidues, all varieties of amino acids, L or D conformations as well asmodified amino acids, are preferably incorporated during each cycle ofthe synthesis. It should be noted, however, that various parameters,e.g., solvent incompatibility of certain amino acids, may result in amixture which contains peptides lacking certain amino acids. The processshould thus be adjusted as needed, by altering solvents and reactionconditions, to produce the greatest variety of peptides. A peptide usedfor peptide loading as described in Example 5 with murine MHC class IIis ovalbumin₃₂₃₋₃₃₉ (OVA) having the amino acid residue sequenceISQAVHAAHEINEAGR (SEQ ID NO 52). Preferred peptides specific for humanMHC class II haplotypes include the following peptides: 1) Influenzahemagglutinin₃₀₆₋₃₁₈ PKYVKQNTLKLAT (SEQ ID NO 53) that bindsHLA-DRB1*0101; 2) HSP65₂₋₁₂ from M. tuberculosis KTIATDEEARR (SEQ ID NO54) that binds HLA-DRB1*0301; 3) Mitochondrial outer protein (MOMP) ofC. trachomatis QASLALSYRLNMFTP (SEQ ID NO 55) that binds HLA-DRB1*0301;and 4) HLA-A2₃₃₋₄₅ FVRFDSDAASQRM (SEQ ID NO 56) that binds toHLA-DRB1*0401. An extensive listing of known class II-binding peptidesas well as a description of how peptide ligands are defined is presentedby Rammensee et al., Immunogenetics, 41:178-228 (1995). Peptides for usein practicing the methods of this invention are also available throughChiron Mimetopes. In other embodiments, peptide libraries, readilyproduced by one of ordinary skill in the art, are contemplated for usein practicing the methods of this invention with the synthetic antigenpresenting cells as described herein.

5. Loading of Empty MHC Class II Molecules by In Vitro Incubation withPeptides

As shown by the results presented below, the murine MHC class IImolecules along with selected accessory molecules expressed on thesurface of Drosophila cell lines, having been transfected with variouscombinations of plasmids encoding MHC class II and accessory molecules,bind specific peptides in a dose-dependent fashion and are able topresent these peptides to antigen specific T cells resulting in T cellactivation.

For the proliferation and activation assays performed below, Drosophilamelanogaster Schneider 2 (SC2) cells (ATCC Accession Number CRL 10974)were transformed with genes encoding the MHC class II α- and β-chainfrom H2-A^(d) as described in Example 2. For other assays where selectedaccessory cells were expressed in combination with the MHC class IImolecules, the genes for B7.1, B7.2 and ICAM-1, were also introducedinto the MHC gene-containing cells as previously described.

The accessory molecules were expressed at selected concentrations and incombinations as defined below to permit reproducible and consistentactivation of CD4⁺ cells. Following expression, induced by treatmentwith copper sulfate, the concentrations of expressed molecules weremonitored. Selection of cells expressing the desired levels of moleculeswas performed with flow cytometry sorting of cell lines. Table I liststhe Drosophila cell lines expressing murine IA^(d) MHC class IImolecules alone or in combination with various accessory molecules. Thetable also presents a summary of the results of proliferative effects,described further below, as indicated with a “−” for no effect and a “+”for a positive proliferative effect. A summary of the types of cytokinesproduced by each type of synthetic antigen presenting cell is alsoindicated, and described further below, with the additional notationwith arrows of whether a particular cytokine production was up or downregulated.

TABLE 1 Accessory Proliferative Cytokines Molecules Responses ProducedNone − — B7.1 + IL-4, IL-10, IL-2 B7.2 + IL-4, IL-10, IL-2 ICAM-1 − —B7.1 + B7.2 + IL-4, IL-10, IL-2 B7.1 + ICAM-1 + ↑IL-2, ↓IL-4, ↓IL-10B7.2 + ICAM-1 + ↑IL-2, ↓IL-4, ↓IL-10 CD70 + ICAM-1 + IL-2 CD70 − —CD70 + ICAM-1 + + ↑IL-2 B7.2

For assessing the effect of antigen presentation and ultimately T cellresponse, the antigen specific T cells were CD4⁺ cells obtained from Tcell receptor transgenic mice. The transgenic line as used herein wasD01 which expresses a T cell receptor specific for the ovalbumin peptide323-339. The D01 mouse line is obtained from Dr. Dennis Lo. For assayingT cell responsiveness to the recombinant surface-expressed MHC class IImolecules, CD4⁺ cells were first purified from lymph nodes using acombination of techniques designed to eliminate all host APC from theresponder population. Small numbers (5×10⁴) of these highly purifiedCD4⁺ cells were then cultured with the Drosophila cell lines in thepresence or absence of ova peptide.

The effect of peptide presentation in the presence of MHC class II withand without accessory molecules, B7.1, B7.2 or B7.2 plus ICAM-1, wasfirst assessed in a proliferation assay performed as described by Webbet al., Cell, 63:1249 (1990). Briefly, 5×10⁴ CD4⁺ T cells were culturedin microtiter wells with 5×10⁴ Drosophila cells in 300 μl of culturemedium. Eighteen hours prior to harvesting, 100 μl of supernatant wereremoved for cytokine analysis and 1 μCi of ³H-thymidine was added toeach well. At the times indicated (3, 4 or 5 days), the wells wereindividually harvested onto glass fiber filters and counted in a liquidscintillation counter.

As seen in FIG. 2, cell lines expressing MHC class II molecules alonefailed to stimulate T cell proliferative responses as indicated by thelow incorporation of radioactive thymidine; higher counts indicategreater proliferation plotted against the amount of stimulating peptidein μM. However, co-expression of either B7.1 or B7.2 costimulatorymolecules conferred the capacity of MHC class II presented-ova peptideto stimulate strong proliferative responses. For comparison, the resultswith splenic APC (labeled T-S) are shown in FIG. 2.

As shown in FIG. 3, Drosophila cell lines expressing MHC class IImolecules with the adhesion molecule ICAM-1 were nonstimulatory.However, the expression of ICAM-1 together with B7.2 generated asynthetic APC cell line that stimulated proliferative responses at lowerconcentrations of peptide antigen. These results demonstrate thatDrosophila cell lines expressing the murine H2-A^(d) MHC class IImolecules and costimulatory molecules, B7.1, B7.2 and B7.2 plus ICAM-1,are able to activate naive CD4⁺ T cells as measured by an increase inproliferation.

In addition to proliferation, the reproducible activation of CD4⁺ Tcells to produce particular cytokines was desired. Thus, the cytokinesproduced in the cultures described above were also evaluated usingstandard ELISA methods with antibodies specific for the cytokine to bemeasured. The data plotted in FIGS. 4A-4D and 5A-5D indicate the amountof cytokine produced in ng/ml concentrations against the stimulatorymolecules on APC.

The results of these assays indicate that different combinations ofaccessory molecules on Drosophila APC expressing recombinant MHC classII leads to the production of different cytokines as shown in FIGS.4A-4D and FIGS. 5A-5D. For IL-2 production, a Th1 type response, celllines expressing MHC class II with B7.1 or B7.2 were poorly stimulatoryas shown in FIG. 4A. In order to achieve significant levels of IL-2, theDrosophila cell lines required co-expression of ICAM-1, B7 and class II(FIG. 5A). Production of γ-interferon was also poorly induced byDrosophila cell lines expressing B7.1 and B7.2 (FIG. 4D). In contrast tothe results with IL-2, however, the co-expression of ICAM-1 did notrestore γ-IFN production (FIG. 5C). Splenic APC (labeled as T-S) inducedstrong γ-IFN production suggesting that additional molecules regulatethe production of this cytokine.

IL-4 production, a Th2 type response, is generally not observed duringprimary stimulation of naive CD4⁺ T cells. In keeping with theseobservations, splenic APC (T-S) failed to induce significant IL-4 afterculture with DO1 cells and ova-peptide (FIG. 5B). However, Drosophilacells co-expressing class II MHC molecules with either B7.1 or B7.2 orboth induced strong IL-4 production during culture (FIGS. 4B and 5B).Interestingly, the addition of ICAM-1 had an antagonistic influence onthe production of this cytokine (FIG. 5B). IL-10 was similarly regulatedto IL-4 as shown in FIGS. 4D and 5D.

As described above, the results with a cytokine profile of a Th2 typeresponse, rather than a Th1 type response, were obtained with cell linesexpressing B7 and MHC class II. In contrast, co-expression of MHC classII in combination with B7 and ICAM-1 antagonized the production of IL-4and IL-10 while strongly promoting IL-2, thereby inducing a Th1 typeresponse with support the present invention in the ability to drive theactivation of CD4⁺ T cells into desired T cell subsets. These resultsdefinitively show that Drosophila cell lines can be tailored to expressdefined combinations of accessory molecules for use in achieving adesired population of CD4⁺ T cells that produce different patterns ofcytokines. Thus, the compositions and methods described herein validatethe compositions and methods of use of the present invention.

The foregoing is intended to be illustrative of the present invention,but not limiting. Numerous variations and modifications may be effectedwithout departing from the true spirit and scope of the invention.

1. A method of producing an insect synthetic antigen presenting cell(APC) comprising: a) providing a cell lacking a gene encoding at leastone of MHC class II α-chain, MHC class II β-chain, and an accessorymolecule; and b) transforming the cell with an expressible gene for eachof the genes of (a) lacking in the cell, the gene being operably linkedto a promoter in a vector capable of expressing the gene.
 2. A method ofproducing an insect synthetic antigen presenting cell (APC) comprising:a) providing a cell lacking a gene encoding at least one of MHC class IIα-chain, MHC class II β-chain, an accessory molecule and an antigenprocessing assisting molecule; and b) transforming the cell with anexpressible gene for each of the genes of (a) lacking in the cell, thegene being operably linked to a first operable promoter in a vectorcapable of expressing the gene.
 3. A method of altering a CD4⁺ Tcell-mediated immune response to treat a condition in a patientcomprising: a) collecting CD4⁺ T cells from the patient; b) loading theinsect synthetic antigen presenting cell produced by the method of claim1 with a selected antigen; c) contacting the CD4⁺ T cells with theselected antigen loaded insect synthetic antigen presenting cell of step(b) in vitro in a sufficient amount and for a sufficient time, therebyinducing the contacted CD4⁺ T cells to proliferate and differentiateinto activated CD4⁺ T cells; and d) returning the activated CD4⁺ T-cellsto the patient.
 4. The method of claim 3 wherein the condition is anautoimmune disease.
 5. The method of claim 4 wherein the autoimmunedisease is selected from the group consisting of diabetes, multiplesclerosis, autoimmune thyroiditis, systemic lupus erythromatosus,mysasthenia gravis, Crohn's disease and inflammatory bowel disease. 6.The method of claim 4 wherein the condition is represented by apatient-specific cytokine profile that is produced by a CD4⁺ Th1 typeresponse.
 7. The method of claim 6 wherein the patient-specific cytokineprofile comprises the cytokine selected from the group consisting ofinterleukin-2, interferon-γ and tumor necrosis factor.
 8. The method ofclaim 3 wherein the condition is an allergy.
 9. The method of claim 8wherein the allergy is selected from the group consisting of asthma andcontact sensitivity.
 10. The method of claim 8 wherein the condition isrepresented by a patient-specific cytokine profile that is produced by aCD4⁺ Th2 type response.
 11. The method of claim 10 wherein thepatient-specific cytokine profile comprises the cytokine selected fromthe group consisting of interleukin-4 and interleukin-10.